Genetically engineered microbial strains including prototheca lipid pathway genes

ABSTRACT

Genetically engineered microbial, e.g.,  Prototheca,  cells provide microbial oil useful as a food additive and a source of renewable fuels and industrial chemicals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/688,025, filed Nov. 28, 2012, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/564,247,filed Nov. 28, 2011, U.S. Provisional Patent Application No. 61/581,538,filed Dec. 29, 2011, and U.S. Provisional Patent Application No.61/674,251, filed Jul. 20, 2012. Each of these applications isincorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing.

FIELD OF THE INVENTION

In certain embodiments genetically engineered microbes, e.g., Protothecastrains are provided that show improved oil production and so find usesin the fields of chemistry, food production, fuel and industrialchemicals production, microbiology, and molecular biology.

DESCRIPTION OF RELATED DISCLOSURES

Microalgae, including genetically engineered microalgae, have beenidentified as important new sources of oil for use in food and fuels.See PCT Pub. Nos. WO 2008/151149, WO 2009/126843, and WO 2010/045368,each of which is incorporated herein by reference in its entirety forall purposes. While Chlorella strains have been the focus of much effortin developing microbial oil production methods, more recently strains ofthe genus Prototheca have been identified as also having promise as anew source of microbial oils, including tailored oils for specificapplications. See PCT Pub. Nos. WO 2010/063031, WO 2010/063032, and WO2011/150410and WO 2012/106560, each of which is incorporated herein byreference in its entirety for all purposes.

SUMMARY

In certain embodiments, the invention provides a recombinant (e.g.,isolated) nucleic acid comprising a coding sequence that encodes aPrototheca lipid biosynthesis protein or portion thereof. In someembodiments, provided is a recombinant nucleic acid comprising a codingsequence that encodes a Prototheca lipid biosynthesis protein, providedthat the protein is not ketoacyl acyl carrier protein synthase II,stearoyl acyl carrier protein desaturase, or delta 12 fatty aciddesaturase. In some embodiments the protein is not a ketoacyl acylcarrier protein synthase II protein of SEQ ID NO: 270, a stearol acylcarrier protein desaturase of SEQ ID NO: 271, or a delta 12 fatty aciddesaturase of SEQ ID NOs: 272 or 273. In some embodiments, the codingsequence is in operable linkage with a promoter, an untranslated controlelement, and/or a targeting sequence, such as a plastidial targetingsequence and mitochondrial targeting sequence. The recombinant nucleicacid may be, e.g., a DNA molecule. In certain embodiments, recombinantnucleic acid is an expression vector. The recombinant nucleic acid can,for example, include an expression cassette that encodes an mRNA thatencodes a functional Prototheca lipid biosynthesis enzyme. Alternativelyor in addition, the recombinant nucleic acid can include an expressioncassette that encodes an inhibitory RNA that suppresses expression of aPrototheca lipid biosynthesis gene. In some embodiments, the lipidbiosynthesis protein is a protein in Table 1. In some embodiments, theprotein has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequenceidentity to a protein provided herein or a protein encoded by a geneprovided herein. In some embodiments the protein or the gene encodingthe protein is listed in Table 1.

In some embodiments, the protein encoded by the coding sequence in thenucleic acid contains one or more point mutations, deletions,substitutions, or combinations thereof. In other embodiments, theprotein has at least one point mutation in comparison to a protein inTable 1. In some embodiments, the protein encoded by the coding sequenceis a functional protein. In some embodiments, the protein isdiacylglycerol diacyltransferase (DGAT) having at least one pointmutation. In other embodiments, the recombinant nucleic acid furtherencodes sucrose invertase.

In certain embodiments, also provided is a genetically engineeredmicrobial cell transformed with a recombinant nucleic acid providedherein. In some embodiments, the cell is a microbial, plant, or yeastcell. In particular embodiments, provided is a cell comprising one ormore exogenous gene(s), wherein the exogenous gene is a Prototheca lipidbiosynthesis gene selected from the genes listed in Table 1. Thegenetically engineered microbial cell can, for example, be a cell of thegenus Prototheca or Chlorella. In particular embodiments, the cellcomprises both an endogenous lipid biosynthesis gene and one or moreexogenous Prototheca lipid biosynthesis gene(s) selected from the geneslisted in Table 1. In certain embodiments, the exogenous gene can encodea lipid biosynthesis protein, wherein the amino acid sequence of thelipid biosynthesis protein is identical to the endogenous lipidbiosynthesis protein. For example, the exogenous gene can include anucleotide sequence in which the codons of the nucleotide sequenceencoding the amino acids of the lipid biosynthesis protein have beenaltered, as compared to the codons in the native nucleic acid. Invarious embodiments, the exogenous gene can encode a protein with atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%amino acid identity to the native Prototheca protein. The exogenous genecan, in some embodiments, be in operable linkage with a promoter elementthat is not the native Prototheca promoter, an untranslated controlelement that is not the native Prototheca untranslated control element,and/or a nucleotide sequence encoding a transit peptide that is not thenative Prototheca transit peptide. The transit peptide can, for example,be a plastidial targeting sequence or a mitochondrial targetingsequence. In certain embodiments, the cell has a 23S rRNA sequence withat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotideidentity to SEQ ID NO: 5. In particular embodiments, the cell is aPrototheca cell, wherein the cell has a fatty acid profile that is atleast 10% C8-C14.

In certain embodiments, another aspect of the invention is a method forobtaining microbial oil comprising culturing a genetically engineeredcell, e.g., a Prototheca cell, described above under conditions suchthat oil is produced. In certain embodiments, the microbial oil thusproduced has a fatty acid profile that is at least 10% C8-C14. Theinvention also includes a microbial oil produced by this method.

In particular embodiments, the invention provides genetically engineeredcell, e.g., of the genus Prototheca, wherein the activity of one or moreendogenous lipid biosynthesis gene, selected from the genes listed inTable 1, has been attenuated. In various embodiments, the activity ofthe endogenous gene has been attenuated through chromosomal genedeletion, chromosomal gene insertion, frameshift mutation, pointmutation, and/or inhibitory RNA. The genetically engineered cell can, incertain embodiments, further comprise an exogenous Prototheca lipidbiosynthesis pathway gene selected from the genes listed in Table 1. Inparticular embodiments, one or more allele(s) of an endogenous lipidbiosynthesis gene in the genetically engineered cell is attenuated.

In certain embodiments, one allele of the endogenous lipid biosynthesisgene is replaced, in the genetically engineered cell, with apolynucleotide encoding, e.g., an exogenous Prototheca lipidbiosynthesis pathway gene selected from Table 1 and a selectable marker.In a variation of this embodiment, two or more alleles of the endogenouslipid biosynthesis gene are each replaced with a polynucleotide encodingan exogenous Prototheca lipid biosynthesis pathway gene selected fromTable 1 and a selectable marker. In certain embodiments, the geneticallyengineered cell has a 23S rRNA sequence with at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% nucleotide identity to SEQ ID NO: 5. Inparticular embodiments, the genetically engineered cell is a Protothecacell, wherein the cell has a fatty acid profile that is at least 10%C8-C14. In some embodiments, the cell has a fatty acid profile that isat least at least 50%, 60%, or 70% C12:0. In some embodiments, the cellhas a fatty acid profile that is at least at least 70%, 75%, 80%, 85%,or 90% C 18:1. In certain embodiments, the invention also provides amethod for obtaining microbial oil comprising culturing this geneticallyengineered cell, which may be, e.g., a Prototheca cell, under conditionssuch that oil is produced. In certain embodiments, the microbial oilthus produced has a fatty acid profile that is at least 10% C8-C14. Theinvention also includes a microbial oil produced by this method.

In another aspect, the present invention provides a geneticallyengineered microbial cell, e.g., Prototheca cell, in one or more lipidbiosynthesis genes have been modified to increase or decrease expressionof such one or more genes such that the fatty acid profile of thegenetically engineered strain differs from that of the strain from whichit was derived. In one embodiment, at least two genes have beenmodified. In various embodiments, the genetic modifications include oneor more of the following modifications: (i) attenuation of a gene or itsenzymatic product; and (ii) increased expression of a gene or itsenzymatic product; (iii) altered activity of a gene or its enzymaticproduct.

In various embodiments, the genetically engineered cell has one or moreattenuated genes, wherein the genes attenuated have been attenuated by ameans selected from the group consisting of a homologous recombinationevent and introduction of an exogenous gene that codes for aninterfering RNA. In various embodiments, one or more alleles of a geneare attenuated.

In various embodiments, the genetically engineered cell has one or moreover-expressed genes, wherein the genes over-expressed have beenup-regulated by a means selected from the group consisting ofintroduction of additional copies of said gene into said cell;introduction of new expression control elements for said gene; andalteration of the protein-coding sequence of the gene. In variousembodiments, one or more alleles of a gene are over-expressed.

In various embodiments, the modified genes of the genetically engineeredcell are selected from the group consisting of Prototheca lipidbiosynthesis genes presented in Table 1. In various embodiments, thegenetically engineered cell comprises an exogenous gene selected fromthe group consisting of Prototheca lipid biosynthesis genes presented inTable 1. In various embodiments, the genetically engineered cellcomprises one or more over-expressed alleles of a gene, the geneselected from the group consisting of Prototheca lipid biosynthesisgenes presented in Table 1. In various embodiments, the geneticallyengineered cell has an attenuated gene selected from the groupconsisting of Prototheca lipid biosynthesis genes presented in Table 1.In various embodiments, the genetically engineered cell has one moreattenuated alleles of a gene, the gene selected from the groupconsisting of Prototheca lipid biosynthesis genes presented in Table 1.

In various embodiments, the genetically engineered cell has a fatty acidprofile selected from the group consisting of: 3% to 60% C8:0, 3% to 60%C10:0, 3% to 70% C12:0, 3% to 95% C14:0, 3% to 95% C16:0, 3% to 95%C18:0, 3% to 95% C18:1, 0% to 60% C18:2, 0% to 60% C18:3 or combinationsthereof. In various embodiments, the ratio of C10:0 to C12:0 is at least3:1. In some cases, the ratio of C10:0 to C14:0 is at least 10:1. Invarious embodiments, the ratio of C12:0 to C14:0 is at least 3:1. Invarious embodiments, the genetically engineered cell has a fatty acidprofile of at least 40% saturated fatty acids, of at least 60% saturatedfatty acids, or at least 85% saturated fatty acids.

In another aspect, the present invention provides methods for obtainingmicrobial oil comprising culturing a genetically engineered Protothecacell of the invention under conditions such that oil is produced. Invarious embodiments, the microbial oil has a fatty acid profile selectedfrom the group consisting of: 3% to 40% C8:0, 3% to 60% C10:0, 3% to 70%C12:0, 3% to 95% C14:0, 3% to 95% C16:0, 3% to 95% C18:0, 3% to 95%C18:1, 0% to 60% C18:2, 0% to 60% C18:3 or combinations thereof. Invarious embodiments, the ratio of C10:0 to C12:0 is at least 3:1. Insome cases, the ratio of C10:0 to C14:0 is at least 10:1. In variousembodiments, the ratio of C12:0 to C14:0 is at least 3:1. In variousembodiments, the genetically engineered cell has a fatty acid profile ofat least 40% saturated fatty acids, of at least 60% saturated fattyacids, or at least 85% saturated fatty acids.

In an additional aspect, the present invention provides microbial oilsand foods, fuels, and chemicals containing said oil or a chemicalderived therefrom.

In another aspect, the present invention provides recombinant nucleicacids useful in methods for making genetically modified Prototheca andother cells. The nucleic acids of the invention comprise all or someportion of a Prototheca lipid biosynthesis gene.

In various embodiments, these nucleic acids include expressioncassettes, which consist of a coding sequence and control sequences thatregulate expression of the coding sequence, which may code for an mRNAthat encodes a lipid biosynthesis protein, enzyme, or for an RNAi thatacts to suppress expression of a lipid biosynthesis gene.

In other embodiments, these nucleic acids are expression vectors thatinclude one or more expression cassettes and stably replicate in aPrototheca or other host cell, either by integration into chromosomalDNA of the host cell or as freely replicating vectors.

In other embodiments, these nucleic acids comprise only a portion of aPrototheca lipid biosynthesis gene, which portion may be a portion of acoding sequence, an exon, or a control element. Such nucleic acids areuseful in the construction of expression cassettes for Prototheca andnon-Prototheca host cells, for integration of exogenous DNA intoPrototheca host cells, and for construction of nucleic acids useful forattenuating Prototheca lipid biosynthetic genes by homologousrecombination.

In some embodiments, provided are sequences, compositions, host cells,and methods for overexpression of a lipid biosynthesis gene. In someaspects, the overexpressed lipid biosynthesis gene is one or more ofLEC2, DGAT, ATP:citrate lyase (ACL), malic enzyme, lipase, fattyacyl-CoA reductase, Acyl-CoA Binding Proteins (ACBPs), or Lipoic AcidSynthase (LS1).

These and other aspects and embodiments of the invention are describedin the accompanying drawings, a brief description of which immediatelyfollows, the detailed description of the invention below, and areexemplified in the examples below. Any or all of the features discussedabove and throughout the application can be combined in variousembodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows PstI restriction maps of Prototheca moriformis FADc alleleswith and without a targeted gene disruption, as described in Example 37.

FIG. 2 shows the results of the Southern blot described in Example 37.

DETAILED DESCRIPTION

For the convenience of the reader, this detailed description of theinvention is divided into sections. Section I provides definitions ofterms used herein. Section II provides an overview of the Protothecalipid biosynthesis pathway. Section III describes culturing methods forPrototheca cells of the invention. Section IV describes geneticengineering methods for Prototheca and genetically engineered cells ofthe invention. Section V describes microbial oils provided by theinvention. Section VI describes nucleic acids of the invention.

Section I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

A nucleic acid “active in microalgae” refers to a nucleic acid that isfunctional in microalgae. For example, a promoter that has been used todrive an antibiotic resistance gene to impart antibiotic resistance to atransgenic microalgae is active in microalgae.

“Acyl carrier protein” or “ACP” is a protein that binds a growing acylchain during fatty acid synthesis as a thiol ester at the distal thiolof the 4′-phosphopantetheine moiety and comprises a component of thefatty acid synthase complex.

“Acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acylmoiety covalently attached to coenzyme A through a thiol ester linkageat the distal thiol of the 4′-phosphopantetheine moiety of coenzyme A.

“Allele” refers to one or two or more forms of a gene or genetic locus.Alleles of a gene may share 100% or less nucleotide sequence identity.Gene products encoded by alleles of a gene may share 100% or less aminoacid sequence identity. Overexperession of different alleles of a geneand/or the gene products encoded therein may confer different phenotypesto a genetically engineered organism. Attenuation of different allelesof a gene and/or the gene products encoded therein may confer differentphenotypes to a genetically engineered organism.

“Attenuation of a Gene” refers to (i) genetically engineering a gene sothat it has, relative to a wild-type gene, different control sequencesthat result in decreased amounts of a gene product (RNA including mRNA,inhibitory RNA molecules, and other RNAs, polypeptides); (ii)genetically engineering a cell so that it has, relative to a wild-typecell, fewer or no detectable copies of a gene and decreased amounts ofthe corresponding gene product; and/or (iii) genetically engineering thecoding sequence of a gene to either decrease the stability and/oractivity of the gene product (i.e., if the increase the stability of anRNA gene product, increase translation of an mRNA gene product, and/ordecrease the level of enzymatic activity of a protein encoded by themRNA gene product, i.e., by making the protein less stable or lessactive (which may also be referred to as “attenuation of an EnzymaticProduct”). An “Attenuated Gene Product” is the gene product ofattenuation of a gene by any of the foregoing methods. An “AttenuatedGene” is a gene that has been genetically engineered by one or more ofthe methods described herein that results in decreased amounts of geneproduct. Attenuation of a gene thus results in “Decreased Expression ofa Gene”, “down-regulation of the gene”, or “inactivation of the gene”.

“Axenic” is a culture of an organism substantially free fromcontamination by other living organisms.

“Biomass” is material produced by growth and/or propagation of cells.Biomass may contain cells and/or intracellular contents as well asextracellular material, includes, but is not limited to, compoundssecreted by a cell.

“Catalyst” is an agent, such as a molecule or macromolecular complex,capable of facilitating or promoting a chemical reaction of a reactantto a product without becoming a part of the product. A catalystincreases the rate of a reaction, after which, the catalyst may act onanother reactant to form the product. A catalyst generally lowers theoverall activation energy required for the reaction such that itproceeds more quickly or at a lower temperature. Thus, a reactionequilibrium may be more quickly attained. Examples of catalysts includeenzymes, which are biological catalysts; heat, which is a non-biologicalcatalyst; and metals used in fossil oil refining processes.

“Co-culture”, and variants thereof such as “co-cultivate” and“co-ferment”, refer to the presence of two or more types of cells in thesame bioreactor. The two or more types of cells may both bemicroorganisms, such as microalgae, or may be a microalgal cell culturedwith a different cell type. The culture conditions may be those thatfoster growth and/or propagation of the two or more cell types or thosethat facilitate growth and/or proliferation of one, or a subset, of thetwo or more cells while maintaining cellular growth for the remainder.

“Coding Sequence” refers to that portion of a gene or expressioncassette that encodes the RNA transcribed from that gene or expressioncassette in a cell, specifically that portion of the mRNA that istranslated into the protein encoded by that mRNA. Any non-translatedportions of a gene between translated portions are referred to as“introns”.

“Cofactor” or “co-factor” is any molecule, other than the substrate,required for an enzyme to carry out its enzymatic activity.

“Complementary DNA” or “cDNA” is a DNA copy of mRNA, usually obtained byreverse transcription of messenger RNA (mRNA) or amplification (e.g.,via polymerase chain reaction (“PCR”)).

“Control Sequence” refers to nucleic acid sequences in a gene orexpression cassette that regulate transcription of a coding sequence andso include promoters, enhancers, transcription termination sequences,and translation initiation sequences.

“Cultivated”, and variants thereof such as “cultured” and “fermented”,refer to the intentional fostering of growth (increases in cell size,cellular contents, and/or cellular activity) and/or propagation(increases in cell numbers via mitosis) of one or more cells by use ofselected and/or controlled conditions. The combination of both growthand propagation may be termed proliferation. Examples of selected and/orcontrolled conditions include the use of a defined medium (with knowncharacteristics such as pH, ionic strength, and carbon source),specified temperature, oxygen tension, carbon dioxide levels, and growthin a bioreactor. Cultivate does not refer to the growth or propagationof microorganisms in nature or otherwise without human intervention; forexample, natural growth of an organism that ultimately becomesfossilized to produce geological crude oil is not cultivation.

“Cytolysis” is the lysis of cells in a hypotonic environment. Cytolysisis caused by excessive osmosis, or movement of water, towards the insideof a cell (hyperhydration). The cell cannot withstand the osmoticpressure of the water inside, and so it explodes.

“Delipidated meal” and “delipidated microbial biomass” is microbialbiomass after oil (including lipids) has been extracted or isolated fromit, either through the use of mechanical (i.e., exerted by an expellerpress) or solvent extraction or both. Delipidated meal has a reducedamount of oil/lipids as compared to before the extraction or isolationof oil/lipids from the microbial biomass but does contain some residualoil/lipid.

“Desaturase” are enzymes in the lipid synthesis pathway responsible forthe introduction of double bonds (unsaturation) into the fatty acidchains of fatty acid or triacylglyceride molecules. Examples include butare not limited to stearoyl-Acyl carrier protein desaturase (SAD) andfatty acid desaturase (FAD), also known as fatty acyl desaturase.

“Expression Cassette” refers to a coding sequence and a promoter,optionally in combination with one or more control sequences. Expressioncassettes for enzymes include, for example and without limitation, atranslation initiation control sequence.

“Expression vector” or “expression construct” or “plasmid” or“recombinant DNA construct” refer to a nucleic acid that has beengenerated via human intervention, including by recombinant means ordirect chemical synthesis, with a series of specified nucleic acidelements that permit transcription and/or translation of a particularnucleic acid in a host cell. The expression vector may be part of aplasmid, virus, or nucleic acid fragment. Typically, the expressionvector includes a nucleic acid to be transcribed operably linked to apromoter. Some expression cassettes are expression vectors, butexpression vectors often contain more than one expression cassette, forexample expression cassettes for selectable markers are sometimesincluded in expression vectors for introducing exogenous genes into hostcells. One of skill in the art understands that a “recombinant nucleicacid” that encodes a particular gene, or portion thereof, is isolatedfrom the specific context in which it naturally occurs.

“Exogenous gene” is a nucleic acid that codes for the expression of anRNA and/or protein that has been introduced (“transformed”) into a cell,and is also referred to as a “transgene”. A transformed cell may bereferred to as a recombinant cell, into which additional exogenousgene(s) may be introduced. The exogenous gene may be from a differentspecies (and so heterologous), or from the same species (and sohomologous), relative to the cell being transformed. Thus, an exogenousgene can include a homologous gene that occupies a different location inthe genome of the cell or is under different control, relative to theendogenous copy of the gene. An exogenous gene may be present in morethan one copy in the cell. An exogenous gene may be maintained in a cellas an insertion into the genome (nuclear or plastid) or as an episomalmolecule.

“Exogenously provided” refers to a molecule provided to the culturemedia of a cell culture.

“Expeller pressing” is a mechanical method for extracting oil from rawmaterials such as soybeans and rapeseed. An expeller press is a screwtype machine, which presses material through a caged barrel-like cavity.Raw materials enter one side of the press and spent cake exits the otherside while oil seeps out between the bars in the cage and is collected.The machine uses friction and continuous pressure from the screw drivesto move and compress the raw material. The oil seeps through smallopenings that do not allow solids to pass through. As the raw materialis pressed, friction typically causes it to heat up.

“Fatty acids” shall mean free fatty acids, fatty acid salts, or fattyacyl moieties in a glycerolipid.

“Fatty acid modification enzyme” or “fatty acid modifying enzyme” refersto an enzyme that alters the covalent structure of a fatty acid.Examples of fatty acid modification enzymes include lipase, fattyacyl-CoA/aldehyde reductase, fatty acyl-CoA reductase, fatty aldehydereductase, fatty aldehyde decarbonylase.

“Fatty acid profile” refers to the distribution of fatty acids in a cellor oil derived from a cell in terms of chain length and/or saturationpattern. In this context the saturation pattern can comprise a measureof saturated versus unsaturated acid or a more detailed analysis of thedistribution of the positions of double bonds in the various fatty acidsof a cell. The fatty acid profile in be readily determined, for exampleby using gas chromatography. In one method, the fatty acids of thetriacylglycerol are converted into a fatty acid methyl ester (FAME)using well known methods. The FAME molecules are then detected by gaschromatography. For example, a separate peak is observed for a fattyacid of 14 carbon atoms with no unsaturation (C14:0) compared to anyother fatty acid such as C14:1. The peak area for each class of FAMEdetermined using GC-FID is proportional to the weight percentages of thefatty acids. Unless specified otherwise, the fatty acid profile isexpressed as a weight percent of the total fatty acid content. Whenreferring to fatty acid profiles, “at least 4% C8-C14” means that atleast 4% by weight of the total fatty acids in a cell or in an extractedglycerolipid composition have a chain length that includes 8, 10, 12 or14 carbon atoms.

“Fatty acid synthesis enzyme” refers to an enzyme that alters the chainlength, saturation, or functional group modification of a fatty acid, orcan otherwise lead to an altered fatty acid profile in a cell. Examplesof fatty acid synthesis enzymes include fatty acyl-ACP thioesterase,desaturase, including stearoyl acyl carrier protein desaturase (SAD) andfatty acyl destaurase (FAD), fatty acyl hydroxylase, and β-keto-acyl-ACPsynthase.

“Fatty acyl-ACP thioesterase” is an enzyme that catalyzes the cleavageof a fatty acid from an acyl carrier protein (ACP) during fatty acidsynthesis.

“Fatty acyl-CoA/aldehyde reductase” is an enzyme that catalyzes thereduction of an acyl-CoA molecule to a primary alcohol.

“Fatty acyl-CoA reductase” is an enzyme that catalyzes the reduction ofan acyl-CoA molecule to an aldehyde.

“Fatty aldehyde decarbonylase” is an enzyme that catalyzes theconversion of a fatty aldehyde to an alkane.

“Fatty aldehyde reductase” is an enzyme that catalyzes the reduction ofan aldehyde to a primary alcohol.

“Fixed carbon source” is a molecule(s) containing carbon, typically anorganic molecule, that is present at ambient temperature and pressure insolid or liquid form in a culture media that may be utilized by amicroorganism cultured therein.

“Functional protein” refers to a protein whose its activity has beenretained even though it may be attenuated.

“Genetically engineered”, “genetically engineer”, and “geneticengineering” refers to alteration of the DNA and/or RNA of a living cellby human intervention. Typically, the alteration is mediated by theintroduction of one or more expression vectors, but in some instances,functionally equivalent alterations may be achieved by mutagenesisalone.

“Glycerolipid” refers to a glycerol molecule esterified at the sn-1,sn-2 or sn-3 position of the glycerol with one or more phosphate, fattyacid, phosphoserine, phosphocholine, phosphoinositol, orphosphoethanolamine, or other moieties covalently attached to theglycerol backbone. Examples of glycerolipids include triacylglycerides(triglycerides), diacylglycerides (diglycerides), monoacylglycerides(monoglycerides), glycerol-3-phosphate, lysophosphatidic acid,phosphatidic acid, phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, and phosphatidylethanolamine.

“Glycerolipid synthesis enzyme” refers to an enzyme involved in thesynthesis of glycerolipids. Glycerolipid synthesis enzymes function, forexample, to covalently attach acyl groups to a substituted glycerol.Examples of glycerolipid synthesis enzymes include glycerol-3-phosphateacyltransferase, lysophosphatidic acid acyltransferase, diacylglycerolacyltransferase, phospholipid diacylglycerol acyltransferase, andphosphatidic acid phosphatase.

“Glycerophospholipid” is a glycerolipid that at the sn-1, sn-2 or sn-3positions of the glycerol backbone has at least one or more covalentlybound phosphate or a covalently bound phosphate containing moiety, forexample, phosphocholine, phosphoserine, phosphoinositol, andphosphoethanolamine. Glycerophospholipids include phosphoglycerol,lysophosphatidic acid, phosphatidic acid, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, and phosphatidylethanolamine.

“Heterotrophic” as it pertains to culture conditions is culturing in thesubstantial absence of light while utilizing or metabolizing a fixedcarbon source.

“Homogenate” is biomass that has been physically disrupted.

“Hydrogen:carbon ratio” is the ratio of hydrogen atoms to carbon atomsin a molecule on an atom-to-atom basis. The ratio may be used to referto the number of carbon and hydrogen atoms in a hydrocarbon molecule.For example, the hydrocarbon with the highest ratio is methane CH₄(4:1).

“Hydrophobic fraction” is the portion, or fraction, of a material thatis more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

“Increase lipid yield” refers to an increase in the productivity of amicrobial culture by, for example, increasing dry weight of cells perliter of culture, increasing the percentage of cells that constitutelipid, or increasing the overall amount of lipid per liter of culturevolume per unit time.

“Inducible promoter” is a promoter that mediates transcription of anoperably linked gene in response to a particular stimulus. Examples ofsuch promoters may be promoter sequences that are induced in conditionsof changing pH or nitrogen levels.

“In operable linkage” is a functional linkage between two nucleic acidsequences, such a control sequence (typically a promoter) and the linkedsequence (typically a sequence that encodes a protein, also called acoding sequence). A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

“In situ” means “in place” or “in its original position”.

“Limiting concentration of a nutrient” is a concentration of a compoundin a culture that limits the propagation of a cultured organism. A“non-limiting concentration of a nutrient” is a concentration thatsupports maximal propagation during a given culture period. Thus, thenumber of cells produced during a given culture period is lower in thepresence of a limiting concentration of a nutrient than when thenutrient is non-limiting. A nutrient is said to be “in excess” in aculture, when the nutrient is present at a concentration greater thanthat which supports maximal propagation.

“Lipase” is a water-soluble enzyme that catalyzes the hydrolysis ofester bonds in water-insoluble, lipid substrates. Lipases catalyze thehydrolysis of lipids into glycerols and fatty acids.

“Lipids” are a class of molecules that are soluble in nonpolar solvents(such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties, because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). As used herein, the term “triacylglycerides” and“triglycerides” are interchangeable. “Fats” and “oils” are a subgroup oflipids called “triacylglycerides.” “Oil,” as distinguished from “fat”refers to triacylglycerides that are generally liquid at ordinary roomtemperature and pressure. Fatty acids are conventionally named by thenotation that recites number of carbon atoms and the number of doublebonds separated by a colon. For example oleic acid can be referred to asC18:1 and capric acid can be referred to as C10:0.

“Lipid biosynthesis pathway” or “lipid biosynthetic pathway” or “lipidmetabolic pathway” or “lipid pathway” refers to the synthesis ordegradation of lipids. Thus enzymes of the lipid biosynthesis pathway(e.g. lipid pathway enzyme) include fatty acid synthesis enzymes, fattyacid modification enzymes, and glycerolipid synthesis enzymes, as wellas proteins (e.g. lipid pathway protein) that affect lipid metabolism,i.e., either lipid modification or degradation, and any proteins thatchemically modify lipids, as well as carrier proteins. Lipidbiosynthesis proteins also include transcription factors and kinasesthat are involved in lipid metabolism.

“Lipid biosynthesis gene” is any gene that encodes a protein that isinvolved in lipid metabolism, either in lipid synthesis, modification,or degradation, and any protein that chemically modifies lipidsincluding carrier proteins.

“Lipid pathway enzyme” is any enzyme that plays a role in lipidmetabolism, i.e., either lipid synthesis, modification, or degradation,and any proteins that chemically modify lipids, as well as carrierproteins.

“Lysate” is a solution containing the contents of lysed cells.

“Lysis” is the breakage of the plasma membrane and optionally the cellwall of a biological organism sufficient to release at least someintracellular content, often by mechanical, viral or osmotic mechanismsthat compromise its integrity.

“Lysing” is disrupting the cellular membrane and optionally the cellwall of a biological organism or cell sufficient to release at leastsome intracellular content.

“Microalgae” is a eukarytotic microbial organism that contains achloroplast or plastid, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae include unicellular organisms that separate from sister cellsshortly after cell division, such as Chlamydomonas, as well as microbessuch as, for example, Volvox, which is a simple multicellularphotosynthetic microbe of two distinct cell types. Microalgae includecells such as Chlorella, Dunaliella, and Prototheca. Microalgae alsoinclude other microbial photosynthetic organisms that exhibit cell-celladhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae alsoinclude obligate heterotrophic microorganisms that have lost the abilityto perform photosynthesis, such as certain dinoflagellate algae speciesand species of the genus Prototheca.

“Microorganism” and “microbe” are microscopic unicellular organisms.

“Naturally co-expressed” with reference to two proteins or genes meansthat the proteins or their genes are co-expressed naturally in a tissueor organism from which they are derived, e.g., because the genesencoding the two proteins are under the control of a common regulatorysequence or because they are expressed in response to the same stimulus.

“Overexpression of a Gene” refers to (i) genetically engineering a geneso that it has, relative to a wild-type gene, different controlsequences that result in increased amounts of a gene product (RNA and,if the RNA is an mRNA, the protein encoded by the mRNA) in a cell; (ii)genetically engineering a cell so that it has, relative to a wild-typecell, more copies of a gene and increased amounts of the correspondinggene product; and/or (iii) genetically engineering the coding sequenceof a gene to either increase the stability and/or activity of the geneproduct (i.e., if the increase the stability of an RNA gene product,increase translation of an mRNA gene product, and/or increase the levelof enzymatic activity of a protein encoded by the mRNA gene product,i.e., by making the protein more stable or more active (which may alsobe referred to as “Overexpression of an Enzymatic Product”). An“Overexpressed Gene” is the product of overexpression of a gene by anyof the foregoing methods. Overexpression of a gene thus results in“Increased Expression of a Gene”.

“Promoter” is a nucleic acid control sequence that directs transcriptionof a nucleic acid. As used herein, a promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements, which may belocated as much as several thousand base pairs from the start site oftranscription.

“Prototheca cell” refers to any cell, strain, and species of microalgaeof the genus Prototheca. Illustrative Prototheca cells and strainsinclude, without limitation, those of any of the following species:Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,Prototheca moriformis, and Prototheca zopfii. In one importantembodiment, a Prototheca cell is a cell or strain of Protothecamoriformis. More generally, microalgal cells, strains, and species thatshare greater than 75% sequence identity with the 23s rRNA of Protothecamoriformis or that listed in SEQ ID NO: 5.

“Recombinant” is a cell, nucleic acid, protein or vector that has beenmodified due to the introduction of an exogenous nucleic acid or thealteration of a native nucleic acid. Thus, e.g., recombinant cellsexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes differently than those genesare expressed by a non-recombinant cell. A “recombinant nucleic acid” isa nucleic acid originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,or otherwise is in a form not normally found in nature, including anisolated form, i.e., wherein the nucleic acid is separated from at leastone other component with which the native form of the nucleic acidnaturally occurs. Recombinant nucleic acids may be produced, forexample, to place two or more nucleic acids in operable linkage. Thus,an isolated nucleic acid or an expression vector formed in vitro byligating DNA molecules that are not normally joined in nature, are bothconsidered recombinant for the purposes of this invention. Once arecombinant nucleic acid is made and introduced into a host cell ororganism, it may replicate using the in vivo cellular machinery of thehost cell; however, such nucleic acids, once produced recombinantly,although subsequently replicated intracellularly, are still consideredrecombinant for purposes of this invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid.

The term “replacement” or “replace” or “replaced” when used in referenceto modification of a gene sequence by another refers to the ablation orknockout of an endogenous gene by homologous recombination with anexogenous gene sequence containing suitable flanking regions.

“Inhibitory RNA” refers to RNA that inhibits gene expression. InhibitoryRNA includes double-stranded interfering RNA. Inhibitory RNA includeslong RNA hairpins, which, in some embodiments, are ·200 to 750nucleotides in length, and comprise a coding sequence of the target geneof 50 to 650 nucleotides and its complementary sequence separated bysequence long enough (typically 25 to 200 nucleotides) to allow thecoding sequence and its complementary to form a double-strandedsequence. RNAi also includes microRNAs, which are shorter than long RNAhairpins comprising typically only 19-22 nucleotides of the codingsequence of the target gene and its complement together with flankingsequences to engage the enzymes in the cell that mediate interferencewith gene expression by RNAi.

“Sonication” is a process of disrupting biological materials, such as acell, by use of sound wave energy.

“Sucrose utilization gene” is a gene that, when expressed, aids theability of a cell to utilize sucrose as an energy source. Proteinsencoded by a sucrose utilization gene are referred to herein as “sucroseutilization enzymes” and include sucrose transporters, sucroseinvertases, and hexokinases such as glucokinases and fructokinases.

“Up-regulation of an exogenous gene” refers to (i) geneticallyengineering a gene so that it has, relative to a wild-type gene,different control sequences that result in increased amounts of a geneproduct (RNA and, if the RNA is an mRNA, the protein encoded by themRNA) in a cell; (ii) genetically engineering a cell so that it has,relative to a wild-type cell, more copies of a gene and increasedamounts of the corresponding gene product; and/or (iii) geneticallyengineering the coding sequence of a gene to either increase thestability and/or activity of the gene product (i.e., if the increase thestability of an RNA gene product, increase translation of an mRNA geneproduct, and/or increase the level of enzymatic activity of a proteinencoded by the mRNA gene product, i.e., by making the protein morestable or more active (which may also be referred to as “Up-regulationof an Enzymatic Product”). An “Up-regulated Gene” is the product ofincreased expression of a gene by any of the foregoing methods.Up-regulation of a gene thus results in “Increased Expression of aGene”.

Section II Prototheca Lipid Biosynthesis Pathway

In certain embodiments the present invention provides recombinantPrototheca cells that have been modified to alter the properties and/orproportions of lipids or fatty acids produced. The lipid biosynthesispathway can further, or alternatively, be modified to alter theproperties and/or proportions of various lipid molecules producedthrough enzymatic processing of lipids and intermediates in the lipidbiosynthesis pathway. In various embodiments, the recombinant Protothecacells of the invention have, relative to their untransformedcounterparts, optimized lipid yield per unit volume and/or per unittime, carbon chain length (e.g., for renewable diesel production or forindustrial chemicals applications requiring lipid feedstock), reducednumber of double or triple bonds, optionally to zero, and increasing thehydrogen:carbon ratio of a particular species of lipid or of apopulation of distinct lipid. In other embodiments, the lipids haveincreased number of double bonds.

In particular embodiments, one or more key enzymes that control branchpoints of metabolism of fatty acids and glycerolipids have beenup-regulated or down-regulated to improve lipid production.Up-regulation, or over-expression, of genes may be achieved, forexample, by transforming cells with expression constructs in which agene encoding the enzyme of interest is expressed, e.g., using a strongpromoter and/or enhancer elements that increase transcription. Suchconstructs can include a selectable marker such that the transformantsmay be subjected to selection, which can result in amplification of theconstruct and an increase in the expression level of the encoded enzyme.Down-regulation, or attenuation, of genes may be achieved, for example,by transforming cells with expression cassettes that ablate, throughhomologous recombination, all or a portion of the chromosomally-encodedcorresponding gene. Expression levels of lipid pathway enzymes can alsooptionally be reduced through the use of inhibitory RNA constructs.Optionally, endogenous lipid pathway genes may be modified to alterindividually or in combination their enzymatic specificity, level ofexpression, or cellular localization. The expression cassettes used inup- or down-regulation can replicate by integration into chromosomal DNAof the host cell or as a freely replicating vector.

Genes and gene products of the Prototheca morifomis (UTEX 1435) lipidbiosynthesis pathway are listed in Table 1 and detailed in thesubsections A-K below. Where noted, different alleles of genes areprovided. Typically, modest amino acid changes are seen between the twoproteins, with alleles typically being 0-2% polymorphic in exons, and4-7% polymorphic in introns.

TABLE 1 Prototheca Lipid Biosynthesis Genes NAD-dependentglycerol-3-phosphate dehydrogenase (SEQ ID NO: 204, nucleotide, SEQ IDNO: 205, protein), Fumarate hydratase (SEQ ID NO: 170, nucleotide, SEQID NO: 171, protein), Pyruvate dehydrogenase, Alpha subunit (SEQ ID NO:238, nucleotide, SEQ ID NO: 239, protein) Pyruvate dehydrogenase, Betasubunit (SEQ ID NO: 240, nucleotide, SEQ ID NO: 241, protein) PyruvateDehydrogenase, DLAT E2 subunit (SEQ ID NO: 242, nucleotide, SEQ ID NO:243, protein) Acetate kinase 1 (ACK1) allele 1 (SEQ ID NO: 107,nucleotide, SEQ ID NO: 108, protein), Acetate kinase 1 (ACK1) allele 2(SEQ ID NO: 109, nucleotide, SEQ ID NO: 110, protein), Acetate kinase 2(ACK2) (SEQ ID NO: 111, nucleotide, SEQ ID NO: 112, protein), Lactatedehydrogenase (SEQ ID NO: 117, nucleotide, SEQ ID NO: 118, protein),Phosphate acetyltransferase allele 1 (SEQ ID NO: 113, nucleotide, SEQ IDNO: 114, protein), Phosphate acetyltransferase allele 2 (SEQ ID NO: 115,nucleotide, SEQ ID NO: 116, protein), Lactate dehydrogenase (SEQ ID NO:117, nucleotide, SEQ ID NO: 118, protein) ACCase, Homomeric acetyl-CoAcarboxylase, (SEQ ID NO: 262, nucleotide, SEQ ID NO: 263, protein)Heteromeric acetyl-CoA carboxylase BC subunit allele 1 (SEQ ID NO: 104,nucleotide, SEQ ID NO: 93 protein), Heteromeric acetyl-CoA carboxylaseBC subunit allele 2 (SEQ ID NO: 103, nucleotide, SEQ ID NO: 94 protein),Heteromeric acetyl-CoA carboxylase BCCP subunit allele 1 (SEQ ID NO:101, nucleotide, SEQ ID NO: 95, protein), Heteromeric acetyl-CoAcarboxylase BCCP subunit allele 2 (SEQ ID NO: 102, nucleotide, SEQ IDNO: 96, protein), Acetyl-CoA carboxylase alpha-CT subunit allele 1 (SEQID NO: 92, nucleotide, SEQ ID NO: 97, protein), Acetyl-CoA carboxylasealpha-CT subunit allele 2 (SEQ ID NO: 99, nucleotide, SEQ ID NO: 98,protein), Heteromeric acetyl-CoA carboxylase b-CT subunit, (SEQ ID NO:222, nucleotide, SEQ ID NO: 223, protein) Plastidial ACP allele 2 (SEQID NO: 90, protein) Plastidial Acyl-Carrier Protein (ACP) allele 1 (SEQID NO: 206, nucleotide, SEQ ID NO: 207, protein) MitochondrialAcyl-Carrier Protein (ACP) allele 1 (SEQ ID NO: 64, nucleotide, SEQ IDNO: 65, protein) Mitochondrial Acyl-Carrier Protein (ACP) allele 2 (SEQID NO: 194, nucleotide, SEQ ID NO: 195, protein) Malonyl-CoA:ACPtransacylase (MAT) (SEQ ID NO: 174, nucleotide, SEQ ID NO: 175 protein),Ketoacyl-ACP synthase I allele 1 (KASI allele 1, SEQ ID NO: 69,nucleotide, SEQ ID NO: 70, protein), Ketoacyl-ACP synthase I allele 2(KASI allele 2, SEQ ID NO: 67, nucleotide, SEQ ID NO: 68, protein),Ketoacyl-ACP synthase III (KASIII) (SEQ ID NO: 214, nucleotide, SEQ IDNO: 215, protein), Ketoacyl-ACP reductase (KAR), SEQ ID NO: 258,nucleotide, SEQ ID NO: 259, protein) Ketoacyl-CoA reductase (KCR) (SEQID NO: 184, nucleotide, SEQ ID NO: 185, protein), 3-Hydroxyacyl-ACPdehydrase (HD) (SEQ ID NO: 208, nucleotide, SEQ ID NO: 209, protein),Enoyl-ACP reductase 1 version 1 (ENR1-1) (SEQ ID NO: 218 nucleotide, 220protein) Enoyl-ACP reductase 1 version 2 (ENR1-1) (SEQ ID NO: 219nucleotide, 221 protein) Trans-2-enoyl-CoA reductase (SEQ ID NO: 196,nucleotide, SEQ ID NO: 197, protein), Stearoyl-ACP desaturase 1 allele 1(SAD1 allele 1, SEQ ID NO: 87 nucleotide, SEQ ID NO: 88, protein),Stearoyl-ACP desaturase 1 allele 2 (SAD1 allele 2, SEQ ID NO: 86nucleotide, SEQ ID NO: 85, protein), Fatty acyl-ACP thioesterease A(FATA) allele 1 (SEQ ID NO: 71, nucleotide, SEQ ID NO: 72, protein),Glycerol-3-phosphate acyltransferase (GPAT), (SEQ ID NO: 224,nucleotide, SEQ ID NO: 225, protein), Glycerol-3-phosphateacyltransferase (GPAT) (SEQ ID NO: 186, nucleotide, SEQ ID NO: 187,protein), LPAAT-E, 1-Acyl-sn-glycerol-3-phosphate acyltransferaseisoform E (SEQ ID NO: 81 nucleotide, SEQ ID NO: 82 protein), LPAAT-A,1-Acyl-sn-glycerol-3-phosphate acyltransferase isoform A (SEQ ID NO: 84nucleotide, SEQ ID NO: 83 protein), Posphatidic acid phosphatase (PAP),(SEQ ID NO: 226, nucleotide, SEQ ID NO: 227, protein) Long-chainacyl-CoA ligase (SEQ ID NO: 198, nucleotide, SEQ ID NO: 199, protein),DGAT1-1, Acyl-CoA:Diacylglycerol Acyltransferase 1, Alelle 1 (SEQ ID NO:228, nucleotide, SEQ ID NO: 229, protein) DGAT1-1,Acyl-CoA:Diacylglycerol Acyltransferase 1, Alelle 2 (SEQ ID NO: 230,nucleotide, SEQ ID NO: 231, protein) DGAT2, Acyl-CoA:DiacylglycerolAcyltransferase 2 (SEQ ID NO: 232, nucleotide, SEQ ID NO: 233, protein)Diacylglycerol kinase (DGK)/Sphingosine Kinase (Spik) (SEQ ID NO: 256,nucleotide, SEQ ID NO: 257, protein) Choline kinase (SEQ ID NO: 236,nucleotide, SEQ ID NO: 237, protein) Leafy cotyledon2 (LEC2) (SEQ ID NO:254, nucleotide, SEQ ID NO: 255, protein) ACLA, ATP:Citrate Lyase,Subunit A (SEQ ID NO: 250, nucleotide, SEQ ID NO: 251, protein) ACLB,ATP:Citrate Lyase, Subunit B (SEQ ID NO: 252, nucleotide, SEQ ID NO: 253protein) Malic Enzyme (SEQ ID NO: 248, nucleotide, SEQ ID NO: 249,protein) ACBP1, Acyl-CoA Binding Protein 1 (SEQ ID NO: 244, nucleotide,SEQ ID NO: 245, protein) ACBP2, Acyl-CoA Binding Protein 2 (SEQ ID NO:246, nucleotide, SEQ ID NO: 247, protein) Phosphatidatecytidylyltransferase (SEQ ID NO: 176, nucleotide, SEQ ID NO: 177,protein), Enoyl-CoA hydratase (SEQ ID NO: 192, nucleotide, SEQ ID NO:193, protein), Acyl-CoA oxidase (SEQ ID NO: 190, nucleotide, SEQ ID NO:191, protein), Lineolate FAD3 desaturase allele 1 (SEQ ID NO: 75,nucleotide, SEQ ID NO: 76, protein), Lineolate FAD3 desaturase allele 2(SEQ ID NO: 73, nucleotide, SEQ ID NO: 74, protein), LS, lipoatesynthase, (SEQ ID NO: 260, nucleotide, SEQ ID NO: 261, protein)Glyoxysomal fatty acid beta-oxidation multifunctional protein (SEQ IDNO: 166, nucleotide, SEQ ID NO: 167, protein), Monoglyceride lipase (SEQID NO: 188, nucleotide, SEQ ID NO: 189, protein), Triacylglycerol lipase(SEQ ID NO: 168, nucleotide, SEQ ID NO: 169, protein),Glycerophosphodiester phosphodiesterase (SEQ ID NO: 180, nucleotide, SEQID NO: 181, protein), Membrane bound O-acyl transferasedomain-containing protein (SEQ ID NO: 202, nucleotide, SEQ ID NO: 203,protein), Lipid droplet protein 1, LDP1 (SEQ ID NO: 119, nucleotide, SEQID NO: 120, protein), Succinate semialdehyde dehydrogenase (SEQ ID NO:172, nucleotide, SEQ ID NO: 173, protein), Sterol 14 desaturase (SEQ IDNO: 212, nucleotide, SEQ ID NO: 213, protein) Nitrogen ResponseRegulator (NRR1) (SEQ ID NO: 264, SEQ ID NO: 265) MonoacylglycerolAcyltransferase (MGAT1) (LPLAT-MGAT-like acyltransferase, DAGAT- domaincontaining) (also known in the art as 2-acylglycerol O-acyltransferase)(SEQ ID NO: 266, nucleotide, SEQ ID NO: 267, protein)Cellulase/Endoglucanase (EG1) (SEQ ID NO: 268, nucleotide, SEQ ID NO:269, protein)

A. Acetyl-CoA-Malony-CoA To Acyl-ACP

The early stages of fatty acid synthesis involve the conversion of afixed carbon (e.g., glucose, sucrose, etc.) or other carbon sources intopyruvate. Next, the pyruvate dehydrogenase complex (PDH), comprisingpyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyldehydrogenase, converts the three carbon metabolite pyruvate into thetwo carbon metabolite acetyl-CoA. The acetyl-CoA carboxylase (ACC)complex, utilizing bicarbonate as a substrate, generates the 3-carboncompound malonyl-CoA. Malonyl-CoA:ACP acyltransferase (MAT) thencatalyzes the transfer of a malonyl group from malonyl-CoA to the acylcarrier protein (ACP), thereby generating malonyl-ACP. ACP is used asthe acyl carrier for the various intermediate reactions in fatty acidbiosynthesis. The metabolites acetyl-CoA and malonyl-CoA and the ACPprotein are thus important starting points for fatty acid biosynthesis.

To genetically engineer a microbe for increased production of fattyacids and lipids, recombinant modifications may be made, eitherindividually or in combination to obtain increasedacetyl-CoA/malonyl-CoA/ACP production. For example, to increasemalonyl-CoA production, an expression cassette may be generated and usedto transform a microbe to overexpress polynucleotides encoding one ormore components of the ACC enzyme complex under the control of aconstitutive or regulated promoter. Additional examples of enzymessuitable for up-regulation according to embodiments of the inventioninclude enzymes of the pyruvate dehydrogenase complex (examples, somefrom microalgae, include GenBank Accession Numbers NP_415392; AAA53047;Q1XDM1; and CAF05587). Up-regulation of pyruvate dehydrogenase canincrease production of acetyl-CoA, and thereby increase fatty acidsynthesis.

The acetyl-CoA carboxylase complex catalyzes the initial step in fattyacid synthesis. Accordingly, one or more enzymes comprising this complexmay be up-regulated to increase production of fatty acids (examples,some from microalgae, include GenBank accession numbers BAA94752;AAA75528; AAA81471; YP_537052; YP_536879; NP_045833; and BAA57908).Enzymes of the ACCase complex may include the heteromeric ACCase BCsubunit 1, the heteromeric ACCase BCC subunit, the heteromeric ACCasea-CT subunit, and the heteromeric ACCase b-CT subunit 1.

The ACC exists in two forms, a cytosolic, homomeric (single subunit)form and a heteromeric (multi-subunit) form in the plastid. Both typesof ACC are found in Prototheca (UTEX 1435 strain). A plastid transitpeptide was identified in the homomeric ACCase, indicating it may belocalized to both the plastid and cytoplasm. In some embodiments,provided are sequences and related compositions and methods forexpression of one or more a plastid-targeted homomeric ACC subunit(s).Plastid targeting can be achieved by attaching a leader plastid transitpeptide selected from any of those provided herein, using the endogenousplastid transit peptide on the gene of interest itself, or obtained fromany endogenous plastid-targeted gene in Prototheca containing a plastidtransit peptide, or by using a heterologous plastid transit peptide. Inother embodiments, provided are sequences and related compositions andmethods for overexpression of one or more plastid-targeted, heteromericACC subunit(s). In some embodiments, the ACC overexpression decreasesthe ratio of C18:2 and C18:3 fatty acids by at least 5%, 10%, 15%, 20%,30%, 40%, or 50%.

In some embodiments, ACC and one or more downstream lipid biosynthesisgenes are simultaneously overexpressed. In one embodiment, provided aresequences, compositions, and methods for the simultaneous overexpressionof ACC and DGAT. Starch levels, generally low in Prototheca, can befurther reduced in favor of lipid biosynthesis by overexpressing ACC. Insome embodiments, starch production is attenuated in concert withoverexpression of one or more downstream lipid biosynthesis genes.

In other embodiments, provided are sequences, compositions, and methodsfor fatty acid production by up-regulation of polynucleotides encodingacyl carrier protein (ACP), which carries the growing acyl chains duringfatty acid synthesis. Examples of ACPs, some from microalgae, includeGenBank accession numbers A0T0F8; P51280; NP_849041; YP_874433 as wellas SEQ ID NO: 207. Acyl carrier proteins can differ in subcellularlocalization (SEQ ID NOs: 63-65, SEQ ID NO: 195, and SEQ ID NO: 207).The sequences listed in SEQ ID NO: 194 and SEQ ID NO: 206 provide thecoding sequences of the Prototheca moriformis amino acid sequence givenin SEQ ID NO: 195 and SEQ ID NO: 207, respectively. In one embodiment ofthe present invention, it is advantageous to overexpress a firstexogenous ACP and concomitantly down-regulate a different ACP.

Recombinant modifications may be made to increase the production ofother intermediates in the lipid biosynthesis pathway. For example, toincrease malonyl-ACP production, an expression cassette may be generatedand used to transform a microbe to overexpress polynucleotides given inSEQ ID NO: 174 encoding P. moriformis malonyl-CoA:ACP transacylase (MAT)(SEQ ID NO: 175) active to transfer a malonyl group from malonyl-CoA toacyl carrier protein (ACP). This expression cassette may comprise aconstitutive or inducible promoter active to drive expression of MAT.

In some embodiments, provide are sequences and methods for downregulation of Pyruvate Dehydrogenase Kinase (PDHK), a negative regulatorof pyruvate dehydrogenase complex.

Enzymes that deplete pools of pyruvate or acetyl-CoA for the synthesisof metabolites other than fatty acids may compete with lipidbiosynthesis pathway enzymes for precursor metabolites. Attenuation ofthese competitor enzymes may increase the production of fatty acids orlipids. To genetically engineer a microbe for increased production offatty acids and lipids, recombinant modifications may be made, eitherindividually or in combination to attenuate enzymes that compete formetabolite precursors. For example, to decrease the use of acetyl-CoAfor acetate production, an expression cassette may be generated toablate the gene or genes encoding P. morifomis (UTEX 1435) acetatekinase enzymes (SEQ ID NOs: 107-112). Attenuation of acetate kinase canalso be achieved through the construction and use of expressioncassettes comprising an antisense RNA under the control of aconstitutive or regulated promoter. Additional examples of enzymessuitable for down-regulation according to embodiments of the presentinvention include P. morifomis (UTEX 1435) lactate dehydrogenase (SEQ IDNO: 117, nucleotide, SEQ ID NO: 118, protein), which synthesizes lactatefrom pyruvate or P. morifomis (UTEX 1435) phosphate acetyltransferase(PTA, SEQ ID NOs: 113-116), which catalyzes the conversion of acetyl-CoAto acetylphosphate, a step in the metabolism of acetate.

B. Acyl-ACP to Fatty Acid

A growing acyl-ACP chain is elongated in 2-carbon increments through aset of four enzymatic reactions involving condensation, a firstreduction reaction, dehydration, and a second reduction reaction. Thesereactions are catalyzed by a condensing enzyme (β-ketoacyl-ACP synthase,KAS), a first reductase enzyme (β-ketoacyl-ACP reductase, KAR),adehydrase (β-hydroxyacyl-ACP dehydrase, HR) and a second reductase(enoyl-ACP reductase, ENR).

The initial condensation reaction between malonyl-ACP and acetyl-CoA toproduce a 4-carbon compound is catalyzed by β-ketoacyl-ACP synthase(KAS) III. Successive 2-carbon additions to the elongating acyl-ACPchain, through C16:0, are catalyzed by KAS I. The enzyme KASII performsa 2-carbon extension of C16:0-ACP to C18:0-ACP. Depending on the desiredproperties of fatty acids to be produced, one or more KAS enzymes may beattenuated or over-expressed in the microbe. For example, to engineer amicrobe for increased production of fatty acids with 18 or more carbonatoms, an expression cassette may be generated and used to transform amicrobe to overexpress polynucleotides encoding a KASII enzyme under thecontrol of a constitutive or inducible promoter. Examples of KASIIenzymes suitable for use with embodiments of the present invention arethe Prototheca moriformis KASII enzymes provided in SEQ ID NO: 106 (seeExample 6) and SEQ ID NO: 211. The protein coding sequences for theseenzymes are provided in SEQ ID NO: 105 and SEQ ID NO: 210. Togenetically engineer a microbe for the increased production of short ormid chain fatty acids, an expression cassette may be generated and usedto transform microbes to decrease the expression of KASII, such asthrough targeted knockdown or the use of inhibitory RNA. Optionally, theknockout or knockdown of KASII may be combined with use ofpolynucleotides to transform a microbe to overexpress polynucleotidesencoding either a KASI or KASIII enzyme. An example of a KASIII enzymesuitable for use with embodiments of the present invention is the P.moriformis KASIII enzyme (provided in SEQ ID NO: 215), encoded by thepolynucleotide sequence given in SEQ ID NO: 214. Examples of KASIenzymes suitable for use with embodiments of the present invention arethe P. moriformis KASI enzymes alleles 1 and 2 provided in SEQ ID NO: 70and 68, respectively. The protein coding sequence for these enzymes areprovided in SEQ ID NO: 69 and SEQ ID NO: 67 respectively.

In an additional embodiment, a recombinant cell is engineered tooverexpress a KASI and/or KASIII enzyme in a genetic background whereinKASII enzyme activity has not been attenuated. In still a furtherembodiment, recombinant polynucleotides are generated and used totransform a microbe to ablate or down-regulate a KASI enzyme to increasethe production of fatty acids with greater than 16 carbons. Optionally,the knockout or knockdown of KASI may be combined with the use ofpolynucleotides to transform a microbe to overexpress polynucleotidesencoding KASII.

To genetically engineer a microbe for increased production of specificfatty acids and lipids, recombinant modifications may be made, eitherindividually or in combination to attenuate or overexpress enzymes thatparticipate in fatty acid elongation. Such enzymes includeβ-ketoacyl-ACP reductase (KAR), β-hydroxyacyl-ACP dehydrase (HD), andenoyl-ACP reductase (ER). For example, an expression cassette may begenerated and used to transform a microbe to overexpress polynucleotidesencoding a KAR enzyme, a β-hydroxyacyl-ACP dehydrase, or an enoyl-ACPreductase under the control of a constitutive or inducible promoter.Alternatively, an expression cassette may be generated and used totransform a microbe to overexpress polynucleotides operable to knockoutor attenuate the expression of a KAR enzyme, an HD enzyme, or an ERenzyme. An example of a KAR enzyme suitable for use with embodiments ofthe present invention is the P. moriformis KAR enzyme (provided in SEQID NO: 185), encoded by the polynucleotide sequence given in SEQ ID NO:184. An example of an HD enzyme suitable for use with embodiments of thepresent invention is the P. moriformis HD enzyme (provided in SEQ ID NO:209), encoded by the polynucleotide sequence given in SEQ ID NO: 208.Examples of ER enzymes suitable for use with embodiments of the presentinvention are P. moriformis ER (provided in SEQ ID NO: 201), encoded bythe polynucleotide sequence given in SEQ ID NO: 200 and P. moriformis(UTEX 1435) trans-2-enoyl-CoA reductase (SEQ ID NO: 197, encoded by thepolynucleotide sequence given in SEQ ID NO: 196). According toembodiments of the present invention, it may be advantageous toup-regulate or down-regulate a KAR, HD, or ER enzyme under specificculture conditions, for example during lipid production and/or in agenetic background of a microbe that has been engineered to alteradditional lipid biosynthesis genes or gene products.

Fatty acyl-ACP thioesterase (TE) enzymes terminate elongation byhydrolyzing the acyl-ACP into free fatty acids and ACP. TEs may showspecificity for acyl-ACPs of certain carbon lengths and degree ofsaturation or may be broad TEs, able to cleave acyl-ACP chains ofvarying length and level of saturation. The substrate specificity of TEsis an important contributor to establishing the chain length and degreeof saturation of fatty acids. Depending on the desired length or degreeof saturation of the fatty acid to be produced, one or more genesencoding acyl-ACP thioesterases may be attenuated or over-expressed inthe microbe. For example, an endogenous fatty acyl-ACP thioesterase geneshowing preference for C18:1-ACP (may be knocked out or reduced inexpression while concomitantly a different TE, showing specificity forsaturated C12 and C14-ACPs is overexpressed in the microbe, therebyaltering the population of fatty acids in the microbe. An example an ofacyl-ACP thioesterase suitable for use in embodiments of the presentinvention includes the P. moriformis acyl-ACP thioesterase FATA1(provided in SEQ ID NO: 72), encoded by the polynucleotide sequencegiven in SEQ ID NO:71. Example 5, Example 34, and Example 36 describethe attenuation of a P. moriformis acyl-ACP thioesterase.

C. Unsaturated Fatty Acids and Fatty Acyl Chains

The introduction of carbon-carbon double bonds into a fatty acid, fattyacyl-CoA, or fatty acyl-ACP chains relies on the activity ofdesaturases. Desaturase enzymes may show specificity for the carbonchain length and degree of saturation of their substrates. Specificdesaturases can convert saturated fatty acids or saturated fattyacyl-ACPs to unsaturated fatty acids or unsaturated fatty acyl-ACPs.Other desaturases enzymes may increase the number of carbon-carbondouble bonds of unsaturated fatty acids.

Stearoyl-ACP desaturase (see, e.g., GenBank Accession numbers AAF15308;ABM45911; AAY86086, and SEQ ID Nos: 59-62), for example, catalyzes theconversion of stearoyl-ACP to oleoyl-ACP. Up-regulation of this gene canincrease the proportion of monounsaturated fatty acids produced by acell; whereas down-regulation can reduce the proportion ofmonounsaturates. For illustrative purposes, SADs are responsible for thesynthesis of C18:1 fatty acids from C18:0 precursors.

Additional desaturases are the fatty acyl desaturases (FADs), includingthe phosphatidylglycerol desaturase (FAD4), the plastidial oleatedesaturase (FADE), the plastidial linoleate desaturase (FAD7/FAD8),endoplasmic reticulum oleate desaturase (FAD2), the endoplasmicreticulum linolate desaturase (FAD3), the delta 12 fatty acid desaturase(Δ12 FAD) and the delta 15 fatty acid desaturase (Δ5 FAD). Thesedesaturases also provide modifications with respect to lipid saturation.For illustrative purposes, Δ12 fatty acid desaturases are responsiblefor the synthesis of C18:2 fatty acids from C18:1 precursors and Δ15fatty acid desaturases are responsible for the synthesis of C18:3 fattyacids from C18:2 precursors.

Still additional desaturases, including the palmitate-specificmonogalactosyldiacylglycerol desaturase (FADS), the linoleoyldesaturase, ω-6 fatty acid desaturases, ω-3 fatty acid desaturases, andω-6-oleate desaturases, provide modifications with respect to lipidsaturation. The expression of one or more desaturases, such as ω-6 fattyacid desaturase, ω-3 fatty acid desaturase, or ω-6-oleate desaturase,may be controlled to alter the ratio of unsaturated to saturated fattyacids.

We have found Prototheca to have an extremely compact genome, with verycompact promoter regions and extensive use of bi-directional promoters.For example, Prototheca has a single copy of FAD2 and FAD3 which arecapable of multiple localization. FAD3 encodes two alternate startpositions, one of which encodes a plastid transit peptide and the otherof which does not. In addition FAD3 contains an ER retention signal inits 3′UTR which is shifted away from the COOH carboxy terminal in theplastid-targeted form, and shifted back in the ER-bound form. Thisallows the same gene to go to different sub-cellular locations dependingon the needs of the organism.

Acyl-ACPs synthesized in the plastid are either used directly withinthat organelle to form lipids, including glycerolipids, or exportedoutside the plastid for synthesis of lipids including phospholipids,triacygylcerol, or waxes. Lipid biosynthesis genes may show specificityfor activity in specific subcellular locations.

D. Fatty Acid to Fatty Acyl-CoA

Upon export from the plastid, fatty acids are re-esterified to CoA toform acyl-CoA via the catalytic action of acyl-CoA synthetase. Differentacyl-CoA synthetase enzymes can differ in subcellular localization andshow specificity for fatty acids of differing chain length. An exampleof an acyl-CoA synthetase is the enzyme long chain fatty acyl-CoAsynthetase. Depending on the desired properties of the triacylglycerolto be produced, one or more genes encoding an acyl-CoA synthetase may beattenuated or over-expressed in the microbe.

E. Fatty Acyl-CoA to Triacylglycerol

Triacylglycerides may be formed through three sequentialacyl-CoA-dependent acylations of a sn-glycerol-3-phosphate molecule. Thefirst acylation, the rate-limiting step of glycerolipid synthesis, iscatalyzed by glycerol-3-phosphate acyltransferase (GPAT) to producelyso-phosphatidic acid. The second acylation step is catalyzed by theenzyme acyl-CoA:lyso-phosphatidic acid acyltransferase (LPAAT). Prior tothe third acylation step, the enzyme phosphatidic acid phosphatase (PAP)(or lipins) carries out the removal of the phosphate group fromphosphatidic acid to generate sn-1,2-diacylglycerol (DAG). The finalacyl-CoA-dependent acylation is catalyzed by acyl-CoA: diacylglycerolacyltransferase (DGAT).

Microbes may be genetically engineered for increased production oflipids. For example, to increase the production of TAGs, an expressioncassette may be generated and used to transform a microbe topolynucleotides operable to increase the expression of GPAT. Thisexpression cassette may comprise a constitutive or inducible promoteractive to drive expression of GPAT and may be utilized in the geneticbackground of a strain in which endogenous GPAT activity has beenattenuated. An example of a GPAT suitable for use in an embodiment ofthe present invention is the P. moriformis GPAT, given here as SEQ IDNO: 187, encoded by the sequences given here in SEQ ID NO: 186.

Microbes may be genetically engineered for increased production oftriacylglycerol molecules with desired properties. Certainacyltransferase enzymes, including GPATs, LPAATs, and DGATs maydemonstrate specificity for a subcellular localization or substratespecificity for the length and degree of saturation of the acyl-CoAchain they transfer to the substituted glycerol backbone. Additionally,LPAAT and DGAT enzymes may show substrate specificity for the form ofsubstituted glycerol to which they transfer an acyl-CoA. Depending onthe desired properties of the triacylglyceridesto be produced, one ormore genes encoding GPATs, LPAATs, DGATs, or other acyltranferases maybe attenuated or over-expressed in the microbe. For example, to increasethe production of TAGs with midchain fatty acids esterified at the sn-2position, an expression cassette may be generated and used to transforma microbe to overexpress an LPAAT having specificity for transferringmidchains. This expression cassette may comprise a constitutive orinducible promoter active to drive expression of LPAAT and may beutilized in the genetic background of a strain in which endogenous LPAATactivity has been attenuated. Examples of LPAATs suitable for use inembodiments of the present invention include the P. moriformis LPAAT Eand LPAAT A, given in SEQ ID NO: 82 and SEQ ID NO: 83, encoded by thesequences given in SEQ ID NO: 81 and SEQ ID NO: 84, respectively.

In a similar fashion, to increase production of TAGs, an expressioncassette may be generated and used to transform a microbe to overexpresspolynucleotides encoding a DGAT, active to transfer a acyl-CoA to a DAGmolecule. This expression cassette may comprise a constitutive orinducible promoter active to drive expression of DGAT2. An example of aDGAT suitable for use in the present invention is P. moriformisdiacylglycerol acyltransferase type 2 (DGAT2) (SEQ ID NO: 183, encodedby the sequence given in SEQ ID NO: 182). According to the desiredcharacteristics of the fatty acids or lipids to be produced by therecombinant microbe, it may be advantageous to couple up-regulation of aTE characterized by substrate specificity with one or more GPAT, LPAAT,or DGAT enzymes showing the same substrate specificity.

Additional acyltranferases suitable for use in embodiments of thepresent invention include P. moriformis membrane bound O-acyltransferase domain-containing protein (SEQ ID NO: 203, encoded by thenucleotide sequence provided in SEQ ID NO: 202), P. moriformis putative1-acyl-sn-glycerol-3-phosphate acyltransferase (SEQ ID NO: 179, encodedby the nucleotide sequence provided in SEQ ID NO: 178), and P.moriformis acyl transferase (SEQ ID NO: 217, encoded by the nucleotidesequence provided in SEQ ID NO: 216). The MonoacylglycerolAcyltransferase (MGAT) gene, catalyzed the synthesis of diacyglycerol,and can generally also catalyze the final step in triacylglycerolbiosynthesis. Hence, upregulation of the MGAT gene provided in thisinvention may be desirable.

Alternate lipid pathway enzymes can generate triacylglyceride moleculesthrough a route separate from that above. Enzymes of the fattyacyl-CoA-independent triacylglycerol pathway transfer fatty acyl groupsbetween phosphatidylcholine (PC) moieties employingacyl-lysophosphatidylcholine acyl transferases that may exhibitselective substrate specificity, ultimately transferring them todiacylglycerol.

F. Additional Lipid Molecules

In addition to their incorporation into DAGs and TAGs, fatty acids orfatty acyl molecules may be incorporated into a range of lipid moleculesincluding but not limited to phospholipids, phosphatidylcholine (PC),phosphatidylserine (PS), phosphatidylinositol (PI), sphingolipids (SL),monogalactosyldiacylglycerol, digalactosyldiacylglycerol,liponucleotides, and wax esters. Enzymes that synthesize molecules ofPC, PS, PI, SL, wax esters, liponucleotides, or the galactolipidsmonogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG)may compete with enzymes that lead to or ultimately synthesize DAGs andTAGs for substrates including fatty acids or fatty acyl molecules. Genesencoding proteins involved in the synthesis, utilization, or degradationof PC, PS, PI, SL, monogalactosyldiacylglycerol,digalactosyldiacylglycerol, or wax esters may include diacylglycerolcholinephosphotransferase (DAG-CPT), cytidine diphosphate diacylglycerolsynthase (CTP-DAG synthase), phosphatidylinositol synthase (PIsynthase), choline kinase (CK), phosphatidylinositol-3-kinase(PI3-Kinase), phosphatidylinositol-4-kinase (PI4-Kinase),diacyerolglycerol kinase (DGK), phosphatidylglycerol-3-phosphatephosphatase (PGPP), cholinephosphate cytidylyltransferase (CPCT),phosphatidate cytidylyltransferase, phosphatidylserine decarboxylase(PSD), phospholipase C (PliC), phospholipase D (PliD), sphingolipiddesaturase (SD), monogalactosyldiacylglycerol synthase (MGDG synthase),digalactosyldiacylglycerol synthase (DGDG synthase), ketoacyl-CoAsynthase (KCS), 3-ketoacyl reductase (KR), and wax synthase (WS).Depending on the desired properties of the fatty acids or lipidmolecules to be produced, one or more genes encoding enzymes thatutilize fatty acids or fatty acyl molecules as substrates to producelipid molecules may be attenuated or over-expressed in the microbe.

In one embodiment, provided are sequences, compositions, and methods forinhibition of DGK, which converts DAG to PA. For example, DGK can beinhibited through use of RNAi, hairpin constructs, or double or singleknockouts. In other embodiments, provided are sequences, compositions,and methods for overexpression of epsilon subtype (DGKe). In someaspects, overexpression of DGKe results in selective removal of DAGswith certain acyl groups such as C20:4.

To engineer a microbe for the increased production of triglycerides, itmay be advantageous to attenuate enzymes that support phospholipidsynthesis. For example, to decrease production of the phospholipidcytidine diphosphate (CDP)-diacylglycerol, an expression cassette may begenerated and used to transform a microbe to attenuate phosphatidatecytidylyltransferase, which catalyzes condensation of phosphatidic acidand cytidine triphosphate to produce to CDP-diacylglycerol. An exampleof a phosphatidate cytidylyltransferase suitable for use in anembodiment of the present invention is P. moriformis phosphatidatecytidylyltransferase (SEQ ID NO: 177), encoded by the polynucleotidesequence given by SEQ ID NO: 176. This expression cassette may comprisea constitutive or inducible promoter active to drive down-regulateexpression of the phosphatidate cytidylyltransferase.

Further, additional lipid moieties other than triacylglycerides mayutilize derivations of phosphorylated glycerol as a backbone. Enzymessuch as phosphatidylglycerophosphate synthase (PGP Synthase), involvedin the synthesis of phopholipids may compete with enzymes that providefor triacylglycerols for substrates including phosphorylated forms ofglycerol. Depending on the desired properties of the lipid molecule tobe produced, one or more genes encoding phosphatidylglycerophosphatesynthase may be attenuated or over-expressed in the microbe. LipoateSynthase (LS), also called Lipoyl Synthase or Lipoic Acid Synthase, isgenerally localized to the mitochondria and utilized in the synthesis oflipoic acid. Lipoic acid is an important co-factor and antioxidant.

G. Fatty Acid Degradation

To genetically engineer a microbe for increased production of specificfatty acids and lipids, recombinant modifications may be made, eitherindividually or in combination, to decrease the degradation of fattyacids and lipids. As proteins such as acyl-CoA oxidase, 3-ketoacyl-CoAthiolase, acyl-CoA dehydrogenase, glyoxysomal fatty acid beta-oxidationmultifunctional protein, and enoyl-CoA hydratase are involved in thedegradation of fatty acids, these and other proteins may be attenuatedin the microbe to slow or prevent fatty acid degradation. For example,to engineer a microbe to decrease fatty acid degradation, an expressioncassette may be generated and used to transform a microbe todown-regulate one or more of acyl-CoA oxidase, enoyl-CoA hydratase, andglyoxysomal fatty acid beta-oxidation multifunctional protein, eitherthrough a knockout or knockdown approach. An example of an acyl-CoAoxidase enzyme suitable for use with embodiments of the presentinvention is the Prototheca moriformis acyl-CoA oxidase (provided in SEQID NO: 191), encoded by the polynucleotide sequence given in SEQ ID NO:190). An example of an enoyl-CoA hydratase suitable for use withembodiments of the present invention is the Prototheca moriformisenoyl-CoA hydratase (provided in SEQ ID NO: 193), encoded by thepolynucleotide sequence given in SEQ ID NO: 192. An example of aglyoxysomal fatty acid beta-oxidation multifunctional protein suitablefor use in embodiments of the present invention is the P. moriformis(UTEX 1435) glyoxysomal fatty acid beta-oxidation multifunctionalprotein, presented in SEQ ID NO: 167, encoded by the polynucleotidesequence given in SEQ ID NO: 166. According to the desired chain lengthand degree of saturation of the fatty acids to be produced by therecombinant microbe, it may be advantageous to down-regulate fatty acidor lipid degradation enzymes in the genetic background of a microbe thathas been engineered to alter additional lipid pathway genes or geneproducts.

Long-chain acyl-CoA synthetases (also known in the art as long-chainacyl-CoA ligases) convert free fatty acids into acyl-CoA thioesters.These acyl-CoA thioesters may then be degraded by enzymes involved infatty β-oxidation. To engineer a microbe for decreased fatty aciddegradation, an expression cassette may be generated and used totransform a microbe to down-regulate long-chain acyl-CoA synthetase,either through a knockout or knockdown approach. An example oflong-chain acyl-CoA synthetase suitable for use in an embodiment of thepresent invention is the P. moriformis (UTEX 1435), long-chain acyl-CoAsynthetase presented in SEQ ID NO: 199, encoded by the polynucleotidesequence given in SEQ ID NO: 198. According to the desired chain lengthand degree of saturation of the fatty acids to be produced by therecombinant microbe, it may be advantageous to down-regulate long-chainacyl-CoA synthetase in the genetic background of a microbe that has beenengineered to alter additional lipid pathway genes or gene products.

H. Monoglyceride, Triglyceride, and Lipid Degradation

A strategy to increase the recombinant microbial production oftriglycerides is to prevent or reduce the enzymatic degradation of thesemolecules. Enzymes such as monoglyceride lipase and triacylglycerollipase that hydrolyze triglycerides to fatty acids and glycerol areexamples of proteins that may be attenuated in a microbe to slow orprevent degradation of triglycerides. For example, to engineer a microbeto decrease triglyceride degradation an expression cassette may begenerated and used to transform a microbe to down-regulate monoglyceridelipase or triacylglycerol lipase, either through a knockout or knockdownapproach. An example of a monoglyceride lipase suitable for use inembodiments of the present invention is the P. moriformis (UTEX 1435)monoglyceride lipase presented in SEQ ID NO: 189, encoded by thepolynucleotide sequence given in SEQ ID NO: 188. An example of atriacylglycerol lipase suitable for use in embodiments of the presentinvention is the P. moriformis (UTEX 1435) triacylglycerol lipasepresented in SEQ ID NO: 169, encoded by the polynucleotide sequencegiven in SEQ ID NO: 168. According to embodiments of the presentinvention, it may be advantageous to attenuate one or more lipases underspecific culture conditions, for example during lipid production.

I. Global Regulators

Furthermore, up- and/or down-regulation of genes may be applied toglobal regulators controlling the expression of the genes of the lipidbiosynthetic pathway. Accordingly, one or more global regulators oflipid synthesis may be up- or down-regulated, as appropriate, to inhibitor enhance, respectively, the expression of a plurality of fatty acidsynthetic genes and, ultimately, to increase lipid production. Examplesinclude sterol regulatory element binding proteins (SREBPs), such asSREBP-1a and SREBP-1c (for examples see GenBank accession numbersNP_035610 and Q9WTN3). In one embodiment, a global regulator such as theendogenous LEC2 homolog, may be upregulated to increase lipidproduction. Decoupling or alteration of nitrogen sensing from theprocess of lipid biosynthesis may also be of value (Boyle et al, J.Biol. Chem, May 4, 2012). Also presented in this invention is a NitrogenResponse Regulator, NRR1, a Squamosa Binding protein. In some instanceit may be desirable, for example, to increase the response to nitrogenstarvation by enhancing expression of NRR1.

J. Lipid Droplet Proteins

Eukaryotic cells store triacylglycerol molecules in distinct organelles,often called lipid droplets. Proteins associated with lipid dropletproteins, such as lipid droplet protein 1 (LDP1, SEQ ID NOs: 119-120),are crucial to lipid droplet structure, formation, size, and number. Insome instances, attenuation of lipid droplet proteins results inincreases in lipid droplet size. In other instances, overexpression ofmutated sequences of lipid droplet proteins results in increased lipiddroplet size and number. To genetically engineer a microbe for theproduction of fatty acids and lipids, recombinant modifications may bemade, either individually or in combination to alter the expression oflipid droplet proteins. For example, an expression cassette may begenerated to attenuate or ablate the gene or gene products encodinglipid droplet proteins (SEQ ID NOs: 119-120). Attenuation of geneproducts through the use of RNAi, RNA hairpin, or otherantisense-mediated strategy may be coupled to an inducible orconstitutive promoter. In an additional embodiment, an expressioncassette may be generated to overexpress one or more lipid dropletproteins. Overexpression of lipid droplet proteins may be driven byconstitutive or inducible promoters. P. moriformis (UTEX 1435) does notcontain oleosins, the plant equivalent of algal Lipid Droplet Proteins(LDPs). Instead, algal Lipid Droplet Proteins, such as the LDP1presented here, are suitable for overexpression to alter lipid dropletmorphology.

K. Altering Carbon Metabolism

Numerous enzymatic pathways are involved in metabolizing sugars andmetabolites into intermediates suitable for use in fatty acid or lipidsynthesis or for other cellular pathways. In one embodiment of thepresent invention, it is advantageous to alter the regulation oractivity of enzymes that contribute to production of metabolitesinvolved in lipid synthesis or that utilize the intermediates ormetabolites of lipid synthesis for pathways other than the fatty acidand lipid pathways. The Kreb's cycle is such a metabolic pathway thatconsumes acetyl-CoA to ultimately produce carbon dioxide. Enzymaticparticipants of the Kreb's Cycle include fumarate hydratase (also knownin the art as fumarase). To engineer a microbe for the increasedproduction of specific fatty acids or lipids, an expression cassette maybe generated and used to transform a microbe to attenuate fumaratehydratase, either through a knockout or knockdown approach. An exampleof a fumarate hydratase suitable for use in embodiments of the presentinvention is the P. moriformis (UTEX 1435) fumarate hydratase presentedin SEQ ID NO: 171, encoded by the polynucleotide sequence given in SEQID NO: 170. According to embodiments of the present invention, it may beadvantageous to attenuate fumarate hydratase under specific cultureconditions, for example during lipid production.

An additional example of an enzyme involved in carbon metabolism isNAD-dependent glycerol-3-phosphate dehydrogenase that reversiblyconverts sn-glycerol 3-phosphate to dihydrohyxacetone phosphate, (alsoknown in the art as glycerone phosphate). To increase the level of thetriacylglycerol backbone precursor molecule, the sn-glycerol 3-phosphatemetabolite, an expression cassette may be generated and used totransform a microbe to enhance expression of NAD-dependentglycerol-3-phosphate dehydrogenase. An example of an NAD-dependentglycerol-3-phosphate dehydrogenase suitable for use in embodiments ofthe present invention is the P. moriformis (UTEX 1435) NAD-dependentglycerol-3-phosphate dehydrogenase presented in SEQ ID NO: 205, encodedby the polynucleotide sequence given in SEQ ID NO: 204. According toembodiments of the present invention, it may be advantageous toattenuate NAD-dependent glycerol-3-phosphate dehydrogenase underspecific culture conditions, for example during lipid production. Insome embodiments, it may be advantageous to combine the expression ofseveral pathway enzymes for triacylglycerol production, for example thePAP, G3PDH, GPAT, LPPAT, and DGAT combination.

Other proteins, such as glycerophosphodiester phosphodiesterase,synthesize intermediates of the lipid pathway. Glycerophosphodiesterphosphodiesterase hydrolyses a glycerophosphodiester to form sn-glycerol3-phosphate, which may be used in lipid synthesis. To engineer a microbefor increased production of lipids, an expression cassette may begenerated and used to transform a microbe to overexpress polynucleotidesencoding glycerophosphodiester phosphodiesterase. An example of aglycerophosphodiester phosphodiesterase suitable for use in embodimentsof the present invention is P. moriformis (UTEX 1435)glycerophosphodiester phosphodiesterase (SEQ ID NO: 181), encoded by thethe polynucleotide sequence given in SEQ ID NO: 180. Celluases, such asendoglucanase, are useful for breaking down cellulosic compounds intosugar utilizable by the cell. Provided is an endogenous endoglucanasethat may potentially find uses in secretion to break down cellulosicmaterial, or as a mechanism for loosening cell walls to allow forenhanced lipid droplet formation.

Section III. Cultivation

In certain embodiments, the present invention generally relates tocultivation of microbes, e.g., oleaginous microbes, such as microalgae,including Chlorella and Prototheca species and strains, and yeast,fungi, plant, and bacteria species and strains, for the production ofmicrobial oil (lipids). In particular embodiments, the microbes arerecombinant microbes. The following discussion focuses on Prototheca asan illustrative species. For the convenience of the reader, this sectionis subdivided into subsections. Subsection 1 describes Protothecaspecies and strains and how to identify new Prototheca species andstrains and related microalgae by genomic DNA comparison. Subsection 2describes bioreactors useful for cultivation. Subsection 3 describesmedia for cultivation. Subsection 4 describes oil production inaccordance with illustrative cultivation methods of the invention.

1. Prototheca Species and Strains

Prototheca is a remarkable microorganism for use in the production oflipid, because it can produce high levels of lipid, particularly lipidsuitable for fuel production. The lipid produced by Prototheca hashydrocarbon chains of shorter chain length and a higher degree ofsaturation than that produced by other microalgae. Moreover, Protothecalipid is generally free of pigment (low to undetectable levels ofchlorophyll and certain carotenoids) and in any event contains much lesspigment than lipid from other microalgae. Moreover, recombinantPrototheca cells provided by the invention may be used to produce lipidin greater yield and efficiency, and with reduced cost, relative to theproduction of lipid from other microorganisms. Illustrative Protothecastrains for use in the methods of the invention include Protothecawickerhamii, Prototheca stagnora (including UTEX 327), Protothecaportoricensis, Prototheca moriformis (including UTEX strains 1441,1435), and Prototheca zopfii. In addition, this microalgae growsheterotrophically and may be genetically engineered. Species of thegenus Prototheca are obligate heterotrophs.

Species of Prototheca for use in the invention may be identified byamplification of certain target regions of the genome. For example,identification of a specific Prototheca species or strain may beachieved through amplification and sequencing of nuclear and/orchloroplast DNA using primers and methodology using any region of thegenome, for example using the methods described in Wu et al., Bot. Bull.Acad. Sin. (2001) 42:115-121 Identification of Chlorella spp. isolatesusing ribosomal DNA sequences. Well established methods of phylogeneticanalysis, such as amplification and sequencing of ribosomal internaltranscribed spacer (ITS1 and ITS2 rDNA), 23S rRNA, 18S rRNA, and otherconserved genomic regions may be used by those skilled in the art toidentify species of not only Prototheca, but other hydrocarbon and lipidproducing organisms with similar lipid profiles and productioncapability. For examples of methods of identification and classificationof algae also see for example Genetics, 2005 August; 170(4):1601-10 andRNA, 2005 April; 11(4):361-4.

Thus, genomic DNA comparison may be used to identify suitable species ofmicroalgae to be used in the present invention. Regions of conservedgenomic DNA, such as but not limited to DNA encoding for 23S rRNA, maybe amplified from microalgal species and compared to consensus sequencesin order to screen for microalgal species that are taxonomically relatedto the preferred microalgae used in the present invention. Examples ofsuch DNA sequence comparison for species within the Prototheca genus areshown below. Genomic DNA comparison can also be useful to identifymicroalgal species that have been misidentified in a strain collection.Often a strain collection will identify species of microalgae based onphenotypic and morphological characteristics. The use of thesecharacteristics may lead to miscategorization of the species or thegenus of a microalgae. The use of genomic DNA comparison may be a bettermethod of categorizing microalgae species based on their phylogeneticrelationship.

Microalgae for use in the present invention typically have genomic DNAsequences encoding for 23S rRNA that have at least 99%, least 95%, atleast 90%, or at least 85% nucleotide identity to at least one of thesequences listed in SEQ ID NOs: 1-9.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison may be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score may be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLASTN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

It is understood that the peptide and protein sequences provided hereincan have conservative or non-essential amino acid substitutions that donot have a substantial effect on the function of the peptide.Conservative amino acid substitutions include replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acidsubstitutions, additions, insertions, or deletions.

Other considerations affecting the selection of microorganisms for usein the invention include, in addition to production of suitable lipidsor hydrocarbons for production of oils, fuels, and oleochemicals: (1)high lipid content as a percentage of cell weight; (2) ease of growth;(3) ease of genetic engineering; and (4) ease of biomass processing. Inparticular embodiments, the wild-type or genetically engineeredmicroorganism yields cells that are at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, or at least 70% or morelipid. Preferred organisms grow heterotrophically (on sugars in theabsence of light).

Examples of algae that may be used to practice the present inventioninclude, but are not limited to the following algae: Protothecawickerhamii, Prototheca stagnora, Prototheca portoricensis, Protothecamoriformis, Prototheca zopfii.

2. Bioreactor

Microorganisms are cultured both for purposes of conducting geneticmanipulations and for production of hydrocarbons (e.g., lipids, fattyacids, aldehydes, alcohols, and alkanes). The former type of culture isconducted on a small scale and initially, at least, under conditions inwhich the starting microorganism can grow. Culture for purposes ofhydrocarbon production is usually conducted on a large scale (e.g.,10,000 L, 40,000 L, 100,000 L or larger bioreactors) in a bioreactor.Microalgae, including Prototheca species are typically cultured in themethods of the invention in liquid media within a bioreactor. Typically,the bioreactor does not allow light to enter.

The bioreactor or fermentor is used to culture microalgal cells throughthe various phases of their physiological cycle. Bioreactors offer manyadvantages for use in heterotrophic growth and propagation methods. Toproduce biomass for use in food, microalgae are preferably fermented inlarge quantities in liquid, such as in suspension cultures as anexample. Bioreactors such as steel fermentors can accommodate very largeculture volumes (40,000 liter and greater capacity bioreactors are usedin various embodiments of the invention). Bioreactors also typicallyallow for the control of culture conditions such as temperature, pH,oxygen tension, and carbon dioxide levels. For example, bioreactors aretypically configurable, for example, using ports attached to tubing, toallow gaseous components, like oxygen or nitrogen, to be bubbled througha liquid culture. Other culture parameters, such as the pH of theculture media, the identity and concentration of trace elements, andother media constituents can also be more readily manipulated using abioreactor.

Bioreactors may be configured to flow culture media though thebioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments, for example,media may be infused into the bioreactor after inoculation but beforethe cells reach a desired density. In other instances, a bioreactor isfilled with culture media at the beginning of a culture, and no moreculture media is infused after the culture is inoculated. In otherwords, the microalgal biomass is cultured in an aqueous medium for aperiod of time during which the microalgae reproduce and increase innumber; however, quantities of aqueous culture medium are not flowedthrough the bioreactor throughout the time period. Thus in someembodiments, aqueous culture medium is not flowed through the bioreactorafter inoculation.

Bioreactors equipped with devices such as spinning blades and impellers,rocking mechanisms, stir bars, or means for pressurized gas infusion maybe used to subject microalgal cultures to mixing. Mixing may becontinuous or intermittent. For example, in some embodiments, aturbulent flow regime of gas entry and media entry is not maintained forreproduction of microalgae until a desired increase in number of saidmicroalgae has been achieved.

Bioreactor ports may be used to introduce, or extract, gases, solids,semisolids, and liquids, into the bioreactor chamber containing themicroalgae. While many bioreactors have more than one port (for example,one for media entry, and another for sampling), it is not necessary thatonly one substance enter or leave a port. For example, a port may beused to flow culture media into the bioreactor and later used forsampling, gas entry, gas exit, or other purposes. Preferably, a samplingport may be used repeatedly without altering compromising the axenicnature of the culture. A sampling port may be configured with a valve orother device that allows the flow of sample to be stopped and started orto provide a means of continuous sampling. Bioreactors typically have atleast one port that allows inoculation of a culture, and such a port canalso be used for other purposes such as media or gas entry.

Bioreactors ports allow the gas content of the culture of microalgae tobe manipulated. To illustrate, part of the volume of a bioreactor may begas rather than liquid, and the gas inlets of the bioreactor to allowpumping of gases into the bioreactor. Gases that may be beneficiallypumped into a bioreactor include air, air/CO₂ mixtures, noble gases,such as argon, and other gases. Bioreactors are typically equipped toenable the user to control the rate of entry of a gas into thebioreactor. As noted above, increasing gas flow into a bioreactor may beused to increase mixing of the culture.

Increased gas flow affects the turbidity of the culture as well.Turbulence may be achieved by placing a gas entry port below the levelof the aqueous culture media so that gas entering the bioreactor bubblesto the surface of the culture. One or more gas exit ports allow gas toescape, thereby preventing pressure buildup in the bioreactor.Preferably a gas exit port leads to a “one-way” valve that preventscontaminating microorganisms from entering the bioreactor.

3. Media

Microalgal culture media typically contains components such as a fixednitrogen source, a fixed carbon source, trace elements, optionally abuffer for pH maintenance, and phosphate (typically provided as aphosphate salt). Other components can include salts such as sodiumchloride, particularly for seawater microalgae. Nitrogen sources includeorganic and inorganic nitrogen sources, including, for example, withoutlimitation, molecular nitrogen, nitrate, nitrate salts, ammonia (pure orin salt form, such as, (NH₄)₂SO₄ and NH₄OH), protein, soybean meal,cornsteep liquor, and yeast extract. Examples of trace elements includezinc, boron, cobalt, copper, manganese, and molybdenum in, for example,the respective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂Oand (NH₄)₆Mo₇O₂₄.4H₂O.

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentsmay be difficult to predict. In some cases, certain strains ofmicroorganisms may be unable to grow on a particular growth mediumbecause of the presence of some inhibitory component or the absence ofsome essential nutritional requirement required by the particular strainof microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismsmay be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin, 1 University StationA6700, Austin, Tex., 78712-0183, for its culture collection of algae(UTEX). For example, various fresh water and salt water media includethose described in PCT Pub. No. 2008/151149, incorporated herein byreference.

In a particular example, Proteose Medium is suitable for axeniccultures, and a 1 L volume of the medium (pH ˜6.8) may be prepared byaddition of 1 g of proteose peptone to 1 liter of Bristol Medium.Bristol medium comprises 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3 mMMgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaCl in an aqueoussolution. For 1.5% agar medium, 15 g of agar may be added to 1 L of thesolution. The solution is covered and autoclaved, and then stored at arefrigerated temperature prior to use. Another example is the Protothecaisolation medium (PIM), which comprises 10 g/L postassium hydrogenphthalate (KHP), 0.9g/L sodium hydroxide, 0.1 g/L magnesium sulfate, 0.2g/L potassium hydrogen phosphate, 0.3 g/L ammonium chloride, 10 g/Lglucose 0.001 g/L thiamine hydrochloride, 20 g/L agar, 0.25 g/L5-fluorocytosine, at a pH in the range of 5.0 to 5.2 (see Pore, 1973,App. Microbiology, 26: 648-649). Other suitable media for use with themethods of the invention may be readily identified by consulting the URLidentified above, or by consulting other organizations that maintaincultures of microorganisms, such as SAG, CCAP, or CCALA. SAG refers tothe Culture Collection of Algae at the University of Göttingen(Göttingen, Germany), CCAP refers to the culture collection of algae andprotozoa managed by the Scottish Association for Marine Science(Scotland, United Kingdom), and CCALA refers to the culture collectionof algal laboratory at the Institute of Botany (Reboil, Czech Republic).Additionally, U.S. Pat. No. 5,900,370 describes media formulations andconditions suitable for heterotrophic fermentation of Protothecaspecies.

For oil production, selection of a fixed carbon source is important, asthe cost of the fixed carbon source must be sufficiently low to make oilproduction economical. Thus, while suitable carbon sources include, forexample, acetate, floridoside, fructose, galactose, glucuronic acid,glucose, glycerol, lactose, mannose, N-acetylglucosamine, rhamnose,sucrose, and/or xylose, selection of feedstocks containing thosecompounds is an important aspect of the methods of the invention.Suitable feedstocks useful in accordance with the methods of theinvention include, for example, black liquor, corn starch, depolymerizedcellulosic material, milk whey, molasses, potato, sorghum, sucrose,sugar beet, sugar cane, rice, and wheat. Carbon sources can also beprovided as a mixture, such as a mixture of sucrose and depolymerizedsugar beet pulp. The one or more carbon source(s) may be supplied at aconcentration of at least about 50 μM, at least about 100 μM, at leastabout 500 μM, at least about 5 mM, at least about 50 mM, and at leastabout 500 mM, of one or more exogenously provided fixed carbonsource(s). Highly concentrated carbon sources as feedstock forfermentation are preferred. For example, in some embodiments glucoselevels of at least 300 g/L, at least 400 g/L, at least 500 g/L, or atleast 600 g/L or more of glucose level of the feedstock prior to thecultivation step, is added to a fed batch cultivation, in which thehighly concentrated fixed carbon source is fed to the cells over time asthe cells grow and accumulate lipid. In other embodiments, sucroselevels of at least 500 g/L, at least 600 g/L, at least 700 g/L, at least800 g/L or more of sucrose prior to the cultivation is added to a fedbatch cultivation, in which the highly concentrated fixed carbon sourceis fed to the cells over time as the cells grow and accumulate lipid.Non-limiting examples of highly concentrated fixed carbon source such assucrose include thick cane juice, sugar cane juice, sugar beet juice andmolasses. Carbon sources of particular interest for purposes of thepresent invention include cellulose (in a depolymerized form), glycerol,sucrose, and sorghum, each of which is discussed in more detail below.

In accordance with the present invention, microorganisms may be culturedusing depolymerized cellulosic biomass as a feedstock. Cellulosicbiomass (e.g., stover, such as corn stover) is inexpensive and readilyavailable; however, attempts to use this material as a feedstock foryeast have failed. In particular, such feedstocks have been found to beinhibitory to yeast growth, and yeast cannot use the 5-carbon sugarsproduced from cellulosic materials (e.g., xylose from hemi-cellulose).By contrast, microalgae can grow on processed cellulosic material.Cellulosic materials generally include about 40-60% cellulose; about20-40% hemicellulose; and 10-30% lignin.

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

Cellulosic materials are treated to increase the efficiency with whichthe microbe can utilize the sugar(s) contained within the materials. Theinvention provides novel methods for the treatment of cellulosicmaterials after acid explosion so that the materials are suitable foruse in a heterotrophic culture of microbes (e.g., microalgae andoleaginous yeast). As discussed above, lignocellulosic biomass iscomprised of various fractions, including cellulose, a crystallinepolymer of beta 1,4 linked glucose (a six-carbon sugar), hemicellulose,a more loosely associated polymer predominantly comprised of xylose (afive-carbon sugar) and to a lesser extent mannose, galactose, arabinose,lignin, a complex aromatic polymer comprised of sinapyl alcohol and itsderivatives, and pectins, which are linear chains of an alpha 1,4 linkedpolygalacturonic acid. Because of the polymeric structure of celluloseand hemicellulose, the sugars (e.g., monomeric glucose and xylose) inthem are not in a form that may be efficiently used (metabolized) bymany microbes. For such microbes, further processing of the cellulosicbiomass to generate the monomeric sugars that make up the polymers maybe very helpful to ensuring that the cellulosic materials areefficiently utilized as a feedstock (carbon source).

In another embodiment of the methods of the invention, the carbon sourceis glycerol, including acidulated and non-acidulated glycerol byproductfrom biodiesel transesterification. In one embodiment, the carbon sourceincludes glycerol and at least one other carbon source. In some cases,all of the glycerol and the at least one other fixed carbon source areprovided to the microorganism at the beginning of the fermentation. Insome cases, the glycerol and the at least one other fixed carbon sourceare provided to the microorganism simultaneously at a predeterminedratio. In some cases, the glycerol and the at least one other fixedcarbon source are fed to the microbes at a predetermined rate over thecourse of fermentation.

Some microalgae undergo cell division faster in the presence of glycerolthan in the presence of glucose (see PCT Pub. No. 2008/151149). In theseinstances, two-stage growth processes in which cells are first fedglycerol to rapidly increase cell density, and are then fed glucose toaccumulate lipids can improve the efficiency with which lipids areproduced. The use of the glycerol byproduct of the transesterificationprocess provides significant economic advantages when put back into theproduction process. Other feeding methods are provided as well, such asmixtures of glycerol and glucose. Feeding such mixtures also capturesthe same economic benefits. In addition, the invention provides methodsof feeding alternative sugars to microalgae such as sucrose in variouscombinations with glycerol.

In another embodiment of the methods of the invention, the carbon sourceis invert sugar. Invert sugar is produced by splitting the sucrose intoits monosaccharide components, fructose and glucose. Production ofinvert sugar may be achieved through several methods that are known inthe art. One such method is heating an aqueous solution of sucrose.Often, catalysts are employed in order to accelerate the conversion ofsucrose into invert sugar. These catalysts may be biological, forexample enzymes such as invertases and sucrases may be added to thesucrose to accelerate the hydrolysis reaction to produce invert sugar.Acid is an example of non-biological catalyst, when paired with heat,can accelerate the hydrolysis reaction. Once the invert sugar is made,it is less prone to crystallization compared to sucrose and thus,provides advantages for storage and in fed batch fermentation, which inthe case of heterotrophic cultivation of microbes, including microalgae,there is a need for concentrated carbon source. In one embodiment, thecarbon source is invert sugar, preferably in a concentrated form,preferably at least 800 g/liter, at least 900 g/liter, at least 1000g/liter or at least 1100 g/liter prior to the cultivation step, which isoptionally a fed batch cultivation. The invert sugar, preferably in aconcentrated form, is fed to the cells over time as the cells grow andaccumulate lipid.

In another embodiment of the methods of the invention, the carbon sourceis sucrose, including a complex feedstock containing sucrose, such asthick cane juice from sugar cane processing. Because of the higherdensities of the cultures for heterotrophic oil production, the fixedcarbon source (e.g., sucrose, glucose, etc.) is preferably in aconcentrated form, preferably at least 500 g/liter, at least 600g/liter, at least 700 g/liter or at least 800 g/liter of the fixedcarbon source prior to the cultivation step, which is optionally a fedbatch cultivation in which the material is fed to the cells over time asthe cells grow and accumulate lipid. In the some cases, the carbonsource is sucrose in the form of thick cane juice, preferably in aconcentrated form, preferably at least 60% solids or about 770 g/litersugar, at least 70% solids or about 925 g/liter sugar, or at least 80%solids or about 1125 g/liter sugar prior to the cultivation step, whichis optionally a fed batch cultivation. The concentrated thick cane juiceis fed to the cells over time as the cells grow and accumulate lipid

In one embodiment, the culture medium further includes at least onesucrose utilization enzyme. In some cases, the culture medium includes asucrose invertase. In one embodiment, the sucrose invertase enzyme is asecrectable sucrose invertase enzyme encoded by an exogenous sucroseinvertase gene expressed by the population of microorganisms. Thus, insome cases, as described in more detail in Section IV, below, themicroalgae has been genetically engineered to express a sucroseutilization enzyme, such as a sucrose transporter, a sucrose invertase,a hexokinase, a glucokinase, or a fructokinase.

Complex feedstocks containing sucrose include waste molasses from sugarcane processing; the use of this low-value waste product of sugar caneprocessing can provide significant cost savings in the production ofhydrocarbons and other oils. Another complex feedstock containingsucrose that is useful in the methods of the invention is sorghum,including sorghum syrup and pure sorghum. Sorghum syrup is produced fromthe juice of sweet sorghum cane. Its sugar profile consists of mainlyglucose (dextrose), fructose and sucrose.

Section IV-I. Genetic Engineering Methods and Materials

The present invention provides methods and materials for genenticallymodifying Prototheca cells and recombinant host cells useful in themethods of the present invention, including but not limited torecombinant Prototheca moriformis, Prototheca zopfii, Protothecakrugani, and Prototheca stagnora host cells. The description of thesemethods and materials is divided into subsections for the convenience ofthe reader. In subsection 1, transformation methods are described. Insubsection 2, genetic engineering methods using homologous recombinationare described. In subsection 3, expression vectors and components aredescribed. In subsection 4, selectable markers and components aredescribed.

1. Engineering Methods—Transformation

Cells may be transformed by any suitable technique including, e.g.,biolistics, electroporation (see Maruyama et al. (2004), BiotechnologyTechniques 8:821-826), glass bead transformation and silicon carbidewhisker transformation. Another method that may be used involves formingprotoplasts and using CaCl₂ and polyethylene glycol (PEG) to introducerecombinant DNA into microalgal cells (see Kim et al. (2002), Mar.Biotechnol. 4:63-73, which reports the use of this method for thetransformation of Chlorella ellipsoidea). Co-transformation ofmicroalgae may be used to introduce two distinct vector molecules into acell simultaneously (see for example Jakobiak et al. (2004)Protist;155(4):381-93).

Biolistic methods (see, for example, Sanford, Trends In Biotech. (1988)6:299-302, U.S. Pat. No. 4,945,050; electroporation (Fromm et al., Proc.Nat'l. Acad. Sci. (USA) (1985) 82:5824-5828); use of a laser beam,microinjection or any other method capable of introducing DNA into amicroalgae can also be used for transformation of a Prototheca cell.

2. Engineering Methods—Homologous Recombination

Homologous recombination is the ability of complementary DNA sequencesto align and exchange regions of homology. Transgenic DNA (“donor”)containing sequences homologous to the genomic sequences being targeted(“template”) is introduced into the organism and then undergoesrecombination into the genome at the site of the corresponding genomichomologous sequences. The mechanistic steps of this process, in mostcasees, include: (1) pairing of homologous DNA segments; (2)introduction of double-stranded breaks into the donor DNA molecule; (3)invasion of the template DNA molecule by the free donor DNA endsfollowed by DNA synthesis; and (4) resolution of double-strand breakrepair events that result in final recombination products.

The ability to carry out homologous recombination in a host organism hasmany practical implications for what may be carried out at the moleculargenetic level and is useful in the generation of an oleaginous microbethat can produced tailored oils. By its very nature homologousrecombination is a precise gene targeting event, hence, most transgeniclines generated with the same targeting sequence will be essentiallyidentical in terms of phenotype, necessitating the screening of farfewer transformation events. Homologous recombination also targets geneinsertion events into the host chromosome, resulting in excellentgenetic stability, even in the absence of genetic selection. Becausedifferent chromosomal loci will likey impact gene expression, even fromheterologous promoters/UTRs, homologous recombination may be a method ofquerying loci in an unfamiliar genome environment and to assess theimpact of these environments on gene expression.

Particularly useful genetic engineering applications using homologousrecombination is to co-opt specific host regulatory elements such aspromoters/UTRs to drive heterologous gene expression in a highlyspecific fashion. For example, ablation or knockout of desaturasegenes/gene families with a heterologous gene encoding a selective markermight be expected to increase overall percentage of saturated fattyacids produced in the host cell. Example 3 describes the homologousrecombination targeting constructs and a working example of suchdesaturase gene ablations or knockouts generated in Protothecamoriformis. Another approach to decreasing expression of an endogenousgene is to use an RNA-induced down-regulation or silencing of geneexpression including, but not limited to an RNAi or antisense approach,as well as a dsRNA approach. Antisense, RNAi, RNA hairpin, and dsRNAapproaches are well known in the art and include the introduction of anexpression construct that when expressed as mRNA would lead to theformation of hairpin RNA or an expression construct containing a portionof the target gene that would be transcribed in the antisenseorientation. All four approaches would result in the decreasedexpression of the target gene. Examples 3 and 4 describe expressionconstructs and working examples of the attenuation, or down-regulationof endogenous Prototheca moriformis lipid biosynthesis genes by an RNAhairpin approach.

Because homologous recombination is a precise gene targeting event, itmay be used to precisely modify any nucleotide(s) within a gene orregion of interest, so long as sufficient flanking regions have beenidentified. Therefore, homologous recombination may be used as a meansto modify regulatory sequences impacting gene expression of RNA and/orproteins. It can also be used to modify protein coding regions in aneffort to modify enzyme activites such as substrate specificity,affinities and Km, and thus affecting the desired change in metabolismof the host cell. Homologous recombination provides a powerful means tomanipulate the gost genome resulting in gene targeting, gene conversion,gene deletion, gene duplication, gene inversion and exchanging geneexpression regulatory elements such as promoters, enhancers and 3′UTRs.

Homologous recombination may be achieve by using targeting constructscontaining pieces of endogenous sequences to “target” the gene or regionof interest within the endogenous host cell genome. Such targetingsequences can either be located 5′ of the gene or region of interest, 3′of the gene/region of interest or even flank the gene/region ofinterest. Such targeting constructs may be transformed into the hostcell either as a supercoiled plasmid DNA with additional vectorbackbone, a PCR product with no vector backbone, or as a linearizedmolecule. In some cases, it may be advantageous to first expose thehomologous sequences within the transgenic DNA (donor DNA) with arestriction enzyme. This step can increase the recombination efficiencyand decrease the occurance of undesired events. Other methods ofincreasing recombination efficiency include using PCR to generatetransforming transgenic DNA containing linear ends homologous to thegenomic sequences being targeted.

For purposes of non-limiting illustration, regions of donor DNAsequences that are useful for homologous recombination include the KE858region of DNA in Prototheca moriformis. KE858 is a 1.3 kb, genomicfragment that encompasses part of the coding region for a protein thatshares homology with the transfer RNA (tRNA) family of proteins.Southern blots have shown that the KE858 sequence is present in a singlecopy in the Prototheca moriformis (UTEX 1435) genome. This region andExamples of using this region for homologous recombination targeting hasbeen described in PCT Publication No. WO 2010/063032. Another region ofdonor DNA that is useful is portions of the 6S genomic sequence. The useof this sequence in homologous recombination in Prototheca morifomis isdescribed below in the Examples.

3. Vectors and Vector Components

Vectors for transformation of microorganisms in accordance withembodiments of the present invention may be prepared by known techniquesfamiliar to those skilled in the art in view of the disclosure herein. Avector typically contains one or more genes, in which each gene codesfor the expression of a desired product (the gene product) and isoperably linked to one or more control sequences that regulate geneexpression or target the gene product to a particular location in therecombinant cell. To aid the reader, this subsection is divided intosubsections. Subsection A describes control sequences typicallycontained on vectors as well as novel control sequences provided by thepresent invention. Subsection B describes genes typically contained invectors as well as novel codon optimization methods and genes preparedusing them provided by the invention. Subsection C describes selectablemarkers contained on vectors and provided by the present invention.Subsection D describes methods and procedures used to identify genes.

A. Control Sequences

Control sequences are nucleic acids that regulate the expression of acoding sequence or direct a gene product to a particular location in oroutside a cell. Control sequences that regulate expression include, forexample, promoters that regulate transcription of a coding sequence andterminators that terminate transcription of a coding sequence. Anothercontrol sequence is a 3′ untranslated sequence located at the end of acoding sequence that encodes a polyadenylation signal. Control sequencesthat direct gene products to particular locations include those thatencode signal peptides, which direct the protein to which they areattached to a particular location in or outside the cell.

Thus, an exemplary vector design for expression of an exogenous gene ina microalgae contains a coding sequence for a desired gene product (forexample, a selectable marker, a lipid pathway enzyme, or a sucroseutilization enzyme) in operable linkage with a promoter active inmicroalgae. Alternatively, if the vector does not contain a promoter inoperable linkage with the coding sequence of interest, the codingsequence may be transformed into the cells such that it becomes operablylinked to an endogenous promoter at the point of vector integration. Thepromoterless method of transformation has been proven to work inmicroalgae (see for example Lumbreras, et. al. Plant Journal (1988),14(4), pp. 441-447.

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Illustrative exogenous and/or endogenouspromoters that are active in microalgae (as well as antibioticresistance genes functional in microalgae) are described in PCT Pub. No.2008/151149 and references cited therein

The promoter used to express an exogenous gene may be the promoternaturally linked to that gene or may be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Illustrative promoters include promoterssuch as β-tubulin from Chlamydomonas reinhardtii, used in the Examplesbelow, and viral promoters, such as cauliflower mosaic virus (CMV) andchlorella virus, which have been shown to be active in multiple speciesof microalgae (see for example Plant Cell Rep. 2005 March;23(10-11):727-35; J Microbiol. 2005 August; 43(4):361-5; Mar Biotechnol(NY). 2002 January; 4(1):63-73). Another promoter that is suitable foruse for expression of exogenous genes in Prototheca is the Chlorellasorokiniana glutamate dehydrogenase promoter/5′UTR. Optionally, at least10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used. Illustrative promoters useful forexpression of exogenous genes in Prototheca are listed in the sequencelisting of this application, such as the promoter of the Chlorella HUP1gene (SEQ ID NO:10) and the Chlorella ellipsoidea nitrate reductasepromoter (SEQ ID NO:11). Chlorella virus promoters can also be used toexpress genes in Prototheca, such as sequence numbers 1 to 7 of U.S.Pat. No. 6,395,965. Additional promoters active in Prototheca may befound, for example, in Biochem Biophys Res Commun. 1994 Oct. 14;204(1):187-94; Plant Mol Biol. 1994 October; 26(1):85-93; Virology.2004 Aug. 15; 326(1):150-9; and Virology. 2004 Jan. 5; 318(1):214-23.Other useful promoters are described in detail in the Examples below.

A promoter can generally be characterized as either constitutive orinducible. Constitutive promoters are generally active or function todrive expression at all times (or at certain times in the cell lifecycle) at the same level. Inducible promoters, conversely, are active(or rendered inactive) or are significantly up- or down-regulated onlyin response to a stimulus. Both types of promoters find application inembodiments of the present invention. Inducible promoters useful inembodiments of the present invention include those that mediatetranscription of an operably linked gene in response to a stimulus, suchas an exogenously provided small molecule (e.g., glucose, as in SEQ IDNO:10), temperature (heat or cold), lack of nitrogen in culture media,pH, etc. Suitable promoters can activate transcription of an essentiallysilent gene or upregulate, preferably substantially, transcription of anoperably linked gene that is transcribed at a low level. Examples belowdescribe additional inducible promoters that are useful in Protothecacells.

The termination region, also referred to as a 3′ untranslated region,may be native to the transcriptional initiation region (the promoter),may be native to the DNA sequence of interest, or may be obtainable fromanother source. See, for example, Chen and Orozco, Nucleic Acids Res.(1988), 16:8411.

In an embodiment of the present invention, control sequences thatprovide for the compartmentalized expression of an exogenous enzyme orprotein are utilized to direct the exogenous enzyme or protein to one ormore intracellular ogranelles. Organelles for targeting arechloroplasts, plastids, mitochondria, and endoplasmic reticulum. Anadditional embodiment of the present invention provides recombinantpolynucleotides that enable secretion of a protein outside the cell.

Proteins encoded in the nuclear genome of Prototheca may be targeted tothe plastid using plastid targeting signals. Plastid targeting sequencesendogenous to Chlorella are known, such as genes in the Chlorellanuclear genome that encode proteins that are targeted to the plastid;see for example GenBank Accession numbers AY646197 and AF499684, and inone embodiment, such control sequences are used in the vectors of thepresent invention to target expression of a protein to a Protothecaplastid.

The Examples below describe the use of algal plastid targeting sequencesto target exogenous enzymes and proteins to the correct compartment inthe host cell. Algal plastid targeting sequences were obtained from cDNAlibraries made using Prototheca moriformis and Chlorella protothecodiescells and are described in PCT Publication No. WO 2010/063032.

In another embodiment, the expression of an exogenous enzyme or proteinin Prototheca is targeted to the endoplasmic reticulum. The inclusion ofan appropriate retention or sorting signal in an expression vectorensure that proteins are retained in the endoplasmic reticulum (ER) anddo not go downstream into Golgi. For example, the IMPACTVECTOR1.3vector, from Wageningen UR-Plant Research International, includes thewell known KDEL retention or sorting signal. With this vector, ERretention has a practical advantage in that it has been reported toimprove expression levels 5-fold or more. The main reason for thisappears to be that the ER contains lower concentrations and/or differentproteases responsible for post-translational degradation of expressedproteins than are present in the cytoplasm. ER retention signalsfunctional in green microalgae are known. For example, see Proc. Natl.Acad. Sci. U S A. 2005 Apr. 26; 102(17):6225-30.

In another embodiment of the present invention, a polypeptide istargeted for secretion outside the cell into the culture media. SeeHawkins et al., Current Microbiology Vol. 38 (1999), pp. 335-341 forexamples of secretion signals active in Chlorella that may be used, inaccordance with the methods of the invention, in Prototheca.

B. Genes and Codon Optimization

Typically, a gene includes a promoter, coding sequence, and terminationcontrol sequences. When assembled by recombinant DNA technology, a genemay be termed an expression cassette and may be flanked by restrictionsites for convenient insertion into a vector that is used to introducethe recombinant gene into a host cell. The expression cassette may beflanked by DNA sequences from the genome or other nucleic acid target tofacilitate stable integration of the expression cassette into the genomeby homologous recombination. Alternatively, the vector and itsexpression cassette may remain unintegrated, in which case, the vectortypically includes an origin of replication, which is capable ofproviding for replication of the heterologous vector DNA.

A common gene present on a vector is a gene that codes for a protein,the expression of which allows the recombinant cell containing theprotein to be differentiated from cells that do not express the protein.Such a gene, and its corresponding gene product, is called a selectablemarker. Any of a wide variety of selectable markers may be employed in atransgene construct useful for transforming Prototheca. Examples ofsuitable selectable markers include the G418 resistance gene, thenitrate reductase gene (see Dawson et al. (1997), Current Microbiology35:356-362), the hygromycin phosphotransferase gene (HPT; see Kim et al.(2002), Mar. Biotechnol. 4:63-73), the neomycin phosphotransferase gene,and the ble gene, which confers resistance to phleomycin (Huang et al.(2007), Appl. Microbiol. Biotechnol. 72:197-205). Methods of determiningsensitivity of microalgae to antibiotics are well known. For example,Mol Gen Genet. 1996 Oct. 16; 252(5):572-9.

Other selectable markers that are not antibiotic-based can also beemployed in a transgene construct useful for transforming microalgae ingeneral, including Prototheca species. Genes that confer the ability toutilize certain carbon sources that were previously unable to beutilized by the microalgae can also be used as a selectable marker. Byway of illustration, Prototheca moriformis strains typically growpoorly, if at all, on sucrose. Using a construct containing a sucroseinvertase gene can confer the ability of positive transformants to growon sucrose as a carbon substrate. Additional details on using sucroseutilization as a selectable marker along with other selectable markersare discussed below.

For purposes of the present invention, the expression vector used toprepare a recombinant host cell of the invention will include at leasttwo, and often three, genes, if one of the genes is a selectable marker.For example, a genetically engineered Prototheca of the invention may bemade by transformation with vectors of the invention that comprise, inaddition to a selectable marker, one or more exogenous genes, such as,for example, sucrose invertase gene or acyl ACP-thioesterase gene. Oneor both genes may be expressed using an inducible promoter, which allowsthe relative timing of expression of these genes to be controlled toenhance the lipid yield and conversion to fatty acid esters. Expressionof the two or more exogenous genes may be under control of the sameinducible promoter or under control of different inducible (orconstitutive) promoters. In the latter situation, expression of a firstexogenous gene may be induced for a first period of time (during whichexpression of a second exogenous gene may or may not be induced) andexpression of a second exogenous gene may be induced for a second periodof time (during which expression of a first exogenous gene may or maynot be induced).

In other embodiments, the two or more exogenous genes (in addition toany selectable marker) are: a fatty acyl-ACP thioesterase and a fattyacyl-CoA/aldehyde reductase, the combined action of which yields analcohol product.

Other illustrative vectors of embodiments of the invention that expresstwo or more exogenous genes include those encoding both a sucrosetransporter and a sucrose invertase enzyme and those encoding both aselectable marker and a secreted sucrose invertase. The recombinantPrototheca transformed with either type of vector produce lipids atlower manufacturing cost due to the engineered ability to use sugar cane(and sugar cane-derived sugars) as a carbon source. Insertion of the twoexogenous genes described above may be combined with the disruption ofpolysaccharide biosynthesis through directed and/or random mutagenesis,which steers ever greater carbon flux into lipid production.Individually and in combination, trophic conversion, engineering toalter lipid production and treatment with exogenous enzymes alter thelipid composition produced by a microorganism. The alteration may be achange in the amount of lipids produced, the amount of one or morehydrocarbon species produced relative to other lipids, and/or the typesof lipid species produced in the microorganism. For example, microalgaemay be engineered to produce a higher amount and/or percentage of TAGs.

For optimal expression of a recombinant protein, it is beneficial toemploy coding sequences that produce mRNA with codons preferentiallyused by the host cell to be transformed. Thus, proper expression oftransgenes can require that the codon usage of the transgene matches thespecific codon bias of the organism in which the transgene is beingexpressed. The precise mechanisms underlying this effect are many, butinclude the proper balancing of available aminoacylated tRNA pools withproteins being synthesized in the cell, coupled with more efficienttranslation of the transgenic messenger RNA (mRNA) when this need ismet. When codon usage in the transgene is not optimized, available tRNApools are not sufficient to allow for efficient translation of theheterologous mRNA resulting in ribosomal stalling and termination andpossible instability of the transgenic mRNA.

The present invention provides codon-optimized nucleic acids useful forthe successful expression of recombinant proteins in Prototheca. Codonusage in Prototheca species was analyzed by studying cDNA sequencesisolated from Prototheca moriformis. This analysis represents theinterrogation over 24,000 codons and resulted in Table 2 below.

TABLE 2 Illustrative preferred codon usage in Prototheca strains. AlaGCG 345 (0.36) Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101(0.11) Pro CCG 161 (0.29) GCC 442 (0.46) CCA 49 (0.09) Cys TGT 12 (0.10)CCT 71 (0.13) TGC 105 (0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG226 (0.82) GAC 316 (0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33(0.06) GAA 14 (0.04) AGA 14 (0.02) Phe TTT 89 (0.29) CGG 102 (0.18) TTC216 (0.71) CGA 49 (0.08) Gly GGG 92 (0.12) CGT 51 (0.09) GGA 56 (0.07)CGC 331 (0.57) GGT 76 (0.10) Ser AGT 16 (0.03) GGC 559 (0.71) AGC 123(0.22) His CAT 42 (0.21) TCG 152 (0.28) CAC 154 (0.79) TCA 31 (0.06) IleATA 4 (0.01) TCT 55 (0.10) ATT 30 (0.08) TCC 173 (0.31) ATC 338 (0.91)Thr ACG 184 (0.38) Lys AAG 284 (0.98) ACA 24 (0.05) AAA 7 (0.02) ACT 21(0.05) Leu TTG 26 (0.04) ACC 249 (0.52) TTA 3 (0.00) Val GTG 308 (0.50)CTG 447 (0.61) GTA 9 (0.01) CTA 20 (0.03) GTT 35 (0.06) CTT 45 (0.06)GTC 262 (0.43) CTC 190 (0.26) Trp TGG 107 (1.00) Met ATG 191 (1.00) TyrTAT 10 (0.05) TAC 180 (0.95) Stop TGA/TAG/TAA

In other embodiments, the gene in the recombinant vector has beencodon-optimized with reference to a microalgal strain other than aPrototheca strain. For example, methods of recoding genes for expressionin microalgae are described in U.S. Pat. No. 7,135,290. Additionalinformation for codon optimization is available, e.g., at the codonusage database of GenBank.

While the methods and materials of the invention allow for theintroduction of any exogenous gene into Prototheca, genes relating tosucrose utilization and lipid pathway modification are of particularinterest, as discussed in the following sections.

C. Selectable Markers

Sucrose Utilization

In one embodiment, the recombinant Prototheca cell of the inventionfurther contains one or more exogenous sucrose utilization genes. Invarious embodiments, the one or more genes encode one or more proteinsselected from the group consisting of a fructokinase, a glucokinase, ahexokinase, a sucrose invertase, a sucrose transporter. For example,expression of a sucrose transporter and a sucrose invertase allowsPrototheca to transport sucrose into the cell from the culture media andhydrolyze sucrose to yield glucose and fructose. Optionally, afructokinase may be expressed as well in instances where endogenoushexokinase activity is insufficient for maximum phosphorylation offructose. Examples of suitable sucrose transporters are GenBankaccession numbers CAD91334, CAB92307, and CAA53390. Examples of suitablefructokinases are GenBank accession numbers P26984, P26420 and CAA43322.

In one embodiment, the present invention provides a Prototheca host cellthat secretes a sucrose invertase. Secretion of a sucrose invertaseobviates the need for expression of a transporter that can transportsucrose into the cell. This is because a secreted invertase catalyzesthe conversion of a molecule of sucrose into a molecule of glucose and amolecule of fructose, both of which may be transported and utilized bymicrobes provided by the invention. For example, expression of a sucroseinvertase with a secretion signal (such as that of SEQ ID NO: 12 (fromyeast), SEQ ID NO: 13 (from higher plants), SEQ ID NO: 14 (eukaryoticconsensus secretion signal), and SEQ ID NO: 15 (combination of signalsequence from higher plants and eukaryotic consensus) generatesinvertase activity outside the cell. Expression of such a protein, asenabled by the genetic engineering methodology disclosed herein, allowscells already capable of utilizing extracellular glucose as an energysource to utilize sucrose as an extracellular energy source.

Prototheca species expressing an invertase in media containing sucroseare a preferred microalgal species for the production of oil. Theexpression and extracellular targeting of this fully active proteinallows the resulting host cells to grow on sucrose, whereas theirnon-transformed counterparts cannot. Thus, the present inventionprovides Prototheca recombinant cells with a codon-optimized invertasegene (SEQ ID NO: 16), including but not limited to the yeast invertasegene, integrated into their genome such that the invertase gene isexpressed as assessed by invertase activity and sucrose hydrolysis.

Examples of suitable sucrose invertases include those identified byGenBank accession numbers CAB95010, NP 012104 (SEQ ID NO: 17), andCAA06839. Non-limiting examples of suitable invertases include thosedescribed in PCT Publication No. WO 2010/063032, incorporated herein byreference.

The secretion of an invertase to the culture medium by Prototheca enablethe cells to grow as well on waste molasses from sugar cane processingas they do on pure reagent-grade glucose; the use of this low-valuewaste product of sugar cane processing can provide significant costsavings in the production of lipids and other oils. Thus, the presentinvention provides a microbial culture containing a population ofPrototheca microorganisms, and a culture medium comprising (i) sucroseand (ii) a sucrose invertase enzyme. In various embodiments the sucrosein the culture comes from sorghum, sugar beet, sugar cane, molasses, ordepolymerized cellulosic material (which may optionally contain lignin).In another aspect, the methods and reagents of the inventionsignificantly increase the number and type of feedstocks that may beutilized by recombinant Prototheca. While the microbes exemplified hereare altered such that they can utilize sucrose, the methods and reagentsof the invention may be applied so that feedstocks such as cellulosicsare utilizable by an engineered host microbe of the invention with theability to secrete cellulases, pectinases, isomerases, or the like, suchthat the breakdown products of the enzymatic reactions are no longerjust simply tolerated but rather utilized as a carbon source by thehost.

D. Sequence Determination

A variety of methods may be employed for the identification of genesequences and amino acid sequences of lipid biosynthetic pathway genesand enzymes. Sequences of polynucleotides (e.g., genomic DNA, cDNA, RNA,PCR-amplified nucleotides) may be determined through sequencingtechnologies including but not limited to Sanger sequencing,pyrosequencing, sequencing by synthesis, sequencing by oligonucleotideprobe ligation, and real time sequencing. One skilled in the art maycompare nucleotide sequences to published databases of genomic sequencesor expressed sequences. Where a DNA sequence is determined or disclosed,one skilled in the art may compare segments from published exonsequences, or may assemble exon sequences into a reconstructed sequencethat does not contain intronic sequences. Sequences of polynucleotidesmay also be translated into amino acids, peptides, polypeptides orproteins through a variety of methods including but not limited tomanual translation or computer-automated translation with bioinformaticssoftware commonly known in the art. Comparison methods of sequenced DNA,RNA, amino acids, peptides, or proteins may include but are not limitedto manual evaluation of the sequence or computer-automated sequencecomparison and identification using algorithms such as BLAST (BasicLocal 55 Alignment Search Tool; Altschul, S. E, et al, (1993)/. Mol.Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).

The instant specification teaches partial or complete amino acid andnucleotide sequences encoding one or more particular microbial genes andproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a portion of the disclosed sequencesfor purposes known to those skilled in this art.

Genomic sequencing of P. moriformis (UTEX 1435) was performed usingIllumina HiSeq and paired-end reads were obtained (100 bp reads, ˜450 bpfragment size). Genomic DNA is prepared using standard protocols andfragmented by hydrodynamic shearing. Genomic data using Roche 454technology was also obtained (400 bp fragment size) as were 8 kb matepair libraries. Transcriptome data consisted of Illumina HiSeq pairedend data (100 bp reads, ˜450 bp fragment size). Sequencing reads werequality trimmed and filtered using fastx tools. Genome data wasassembled using Velvet (Zerbino et al, Velvet: algorithms for de novoshort read assembly using de Bruijn graphs, Genome Research, May 2008)using an optimized kmer and other default parameters, and using thePacific Biosciences ALLORA assembler. Annotation was performed using theMAKER pipeline and genes were identified by BLAST against the nrdatabase (NCBI). In addition, a cDNA library was constructed, and 1200cDNAs from this library were sequenced using Sanger sequencing. ThesecDNAs where then annotated and used as initial entry points to findtranscripts from Illumina transcriptomes and to help verify the accuracyof Illumina transcriptome and genome sequence data. Pacific Biosciencestechnology was also utilized to obtain long reads from genomic samples.A given base position is indicated with a code as shown in the tablebelow. In some cases, two representative sequences were constructed,with the two possible bases respectively, as well as correspondingprotein translations for the two alternatives. Such sequences arereferred to as “version 1” and “version 2”.

Base codes A Adenine C Cytosine G Guanine T (or U) Thymine (or Uracil) RA or G Y C or T S G or C W A or T K G or T M A or C B C or G or T D A orG or T H A or C or T V A or C or G N any base . or - gap * stop/nonsensecodon ? unknown amino acid

Section IV-II. Genetically Engineered Prototheca Cells

In a first aspect, the present invention provides a geneticallyengineered Prototheca cell in one or more lipid biosynthesis genes havebeen modified to increase or decrease expression of such one or moregenes such that the fatty acid profile of the genetically engineeredstrain differs from that of the strain from which it was derived. In oneembodiment, at least two genes have been modified. In variousembodiments, the genetic modifications include one or more of thefollowing modifications: (i) attenuation of a gene or its enzymaticproduct; and (ii) increased expression of a gene or its enzymaticproduct; (iii) altered activity of a gene or its enzymatic product.

In various embodiments, the genetically engineered cell has one or moreattenuated genes, wherein the genes attenuated have been attenuated by ameans selected from the group consisting of a homologous recombinationevent and introduction of an exogenous gene that codes for aninterfering RNA. In various embodiments, one or more alleles of a geneare attenuated.

In various embodiments, the genetically engineered cell has one or moreover-expressed genes, wherein the genes over-expressed have beenup-regulated by a means selected from the group consisting ofintroduction of additional copies of said gene into said cell;introduction of new expression control elements for said gene; andalteration of the protein-coding sequence of the gene. In variousembodiments, one or more alleles of a gene are over-expressed.

In various embodiments, the modified genes of the genetically engineeredcell are selected from the group consisting of Prototheca lipidbiosynthesis genes presented in Table 1. In various embodiments, thegenetically engineered cell comprises an exogenous gene selected fromthe group consisting of Prototheca lipid biosynthesis genes presented inTable 1. In various embodiments, the genetically engineered cellcomprises one more over-expressed alleles of a gene, the gene selectedfrom the group consisting of Prototheca lipid biosynthesis genespresented in Table 1. In various embodiments, the genetically engineeredcell has an attenuated gene selected from the group consisting ofPrototheca lipid biosynthesis genes presented in Table 1. In variousembodiments, the genetically engineered cell has one more attenuatedalleles of a gene, the gene selected from the group consisting ofPrototheca lipid biosynthesis genes presented in Table 1.

In various embodiments, the genetically engineered cell has one or moreoverexpressed genes, wherein the expression of the genes have beenincreased by a means selected from the group consisting of introductionof additional copies of said gene into said cell; and introduction ofnew expression control elements for said gene. In various embodiments,the overexpressed gene is an exogenous gene.

In various embodiments, the modified genes of the genetically engineeredcell are selected from the group consisting of Prototheca lipidbiosynthesis genes presented in Table 1.

In various embodiments, the genetically engineered cell has anup-regulated gene selected from the group consisting of Prototheca lipidbiosynthesis genes presented in Table 1. In various embodiments, thegenetically engineered cell has an attenuated gene selected from thegroup consisting of Prototheca lipid biosynthesis genes presented inTable 1. In various embodiments, the genetically engineered cell has afatty acid profile selected from the group consisting of: 3% to 60%C8:0, 3% to 60% C10:0, 3% to 70% C12:0, 3% to 95% C14:0, 3% to 95%C16:0, 3% to 95% C18:0, 3% to 95% C18:1, 3% to 60% C18:2, 1% to 60%C18:3 or combinations thereof. In various embodiments, the ratio ofC10:0 to C12:0 is at least 3:1. In various embodiments, the ratio ofC12:0 to C14:0 is at least 3:1. In some cases, the ratio of C10:0 toC14:0 is at least 10:1. In various embodiments, the geneticallyengineered cell has a fatty acid profile of at least 40% saturated fattyacids, of at least 60% saturated fatty acids, or at least 85% saturatedfatty acids. In various embodiments, the genetically engineered cell hasa fatty acid profile of at least 85% unsaturated fatty acids, of atleast 90% unsaturated fatty acids, of at least 95% unsaturated fattyacids, or at least 97% unsaturated fatty acids.

An embodiment of the present invention also provides recombinantPrototheca cells that have been modified to contain one or moreexogenous genes encoding lipid biosyntheis enzymes such as, for example,a fatty acyl-ACP thioesterase (see Example 5) or a ketoacyl-ACP synthaseII (see Example 6). In some embodiments, genes encoding a fatty acyl-ACPthioesterase and a naturally co-expressed acyl carrier protein aretransformed into a Prototheca cell, optionally with one or more genesencoding other lipid biosynthesis genes. In other embodiments, the ACPand the fatty acyl-ACP thioesterase may have an affinity for one anotherthat imparts an advantage when the two are used together in the microbesand methods of the present invention, irrespective of whether they areor are not naturally co-expressed in a particular tissue or organism.Thus, embodiments of the present invention contemplate both naturallyco-expressed pairs of these enzymes as well as those that share anaffinity for interacting with one another to facilitate cleavage of alength-specific carbon chain from the ACP.

In still other embodiments, an exogenous gene encoding a desaturase istransformed into the Prototheca cell in conjunction with one or moregenes encoding other lipid biosynthesis genes to provide modificationswith respect to lipid saturation. In other embodiments, an endogenousdesaturase gene is overexpressed (e.g., through the introduction ofadditional copies off the gene) in a Prototheca cell. In someembodiments, the desaturase may be selected with reference to a desiredcarbon chain length, such that the desaturase is capable of makinglocation specific modifications within a specified carbon-lengthsubstrate, or substrates having a carbon-length within a specifiedrange. In another embodiment, if the desired fatty acid profile is anincrease in monounsaturates (such as C16:1 and/or C18:1) overexpressionof a SAD or expression of a heterologous SAD may be coupled with thesilencing or inactivation (e.g., through mutation, RNAi, antisense, orknockout of an endogenous desaturase gene, etc.) of a fatty acyldesaturase (FAD) or another desaturase gene.

In other embodiments, the Prototheca cell has been modified to have anattenuated endogenous desaturase gene, wherein the attenuation rendersthe gene or desaturase enzyme inactive. In some cases, the mutatedendogenous desaturase gene is a fatty acid desaturase (FAD). In othercases, the mutated endogenous desaturase gene is a stearoyl acyl carrierprotein desaturase (SAD). Example 4 describes the targeted ablation ofstearoyl-ACP desaturases and delta 12 fatty acid desaturases. Example 4also describes the use of RNA antisense constructs to decrease theexpression of an endogenous desaturase gene. Example

In some cases, it may be advantageous to pair one or more of the geneticengineering techniques in order to achieve a trangenic cell thatproduces the desired fatty acid profile. In one embodiment, a Protothecacell comprises an attenuated endogenous thioestease gene and one or moreexogenous gene. In non-limiting examples, a Prototheca cell with anattenuated endogenous thioesterase gene can also express an exogenousfatty acyl-ACP thioesterase gene and/or a sucrose invertase gene.Example 5 below describes a transgenic Prototheca cell containing atargeted ablation or knockout of an endogenous thioesterase and alsoexpresses a Cuphea wrightii FatB2 C10:0-C14:0 preferring thioesteraseand a sucrose invertase.

In other embodiments, one allele of a Prototheca lipid biosyntheis genehas been attenuated. In additional embodiments, two or more alleles of aPrototheca lipid biosyntheis gene have been attenuated. In additionalembodiments, one or more alleles of different Prototheca lipidbiosyntheis genes have been attenuated. Example 37 below describes thetargeted knockout of multiple alleles of stearoyl-ACP desaturase.Example 34 below describes the targeted knockout of multiple alleles ofacyl-ACP thioesterase. Example 8 below describes the targeted knockoutof acyl-ACP thioesterase and fatty acid desaturase. In some cases, thetargeted knockout of different alleles of a gene may result in differenteffects on fatty acid profiles.

Section V. Microbial Oils

For the production of oil in accordance with the methods of theinvention, it is preferable to culture cells in the dark, as is thecase, for example, when using extremely large (40,000 liter and higher)fermentors that do not allow light to strike the culture. Protothecaspecies are grown and propagated for the production of oil in a mediumcontaining a fixed carbon source and in the absence of light; suchgrowth is known as heterotrophic growth.

As an example, an inoculum of lipid-producing microalgal cells areintroduced into the medium; there is a lag period (lag phase) before thecells begin to propagate. Following the lag period, the propagation rateincreases steadily and enters the log, or exponential, phase. Theexponential phase is in turn followed by a slowing of propagation due todecreases in nutrients such as nitrogen, increases in toxic substances,and quorum sensing mechanisms. After this slowing, propagation stops,and the cells enter a stationary phase or steady growth state, dependingon the particular environment provided to the cells. For obtaining lipidrich biomass, the culture is typically harvested well after then end ofthe exponential phase, which may be terminated early by allowingnitrogen or another key nutrient (other than carbon) to become depleted,forcing the cells to convert the carbon sources, present in excess, tolipid. Culture condition parameters may be manipulated to optimize totaloil production, the combination of lipid species produced, and/orproduction of a specific oil.

As discussed above, a bioreactor or fermentor is used to allow cells toundergo the various phases of their growth cycle. As an example, aninoculum of lipid-producing cells may be introduced into a mediumfollowed by a lag period (lag phase) before the cells begin growth.Following the lag period, the growth rate increases steadily and entersthe log, or exponentia, phase. The exponential phase is in turn followedby a slowing of growth due to decreases in nutrients and/or increases intoxic substances. After this slowing, growth stops, and the cells entera stationary phase or steady state, depending on the particularenvironment provided to the cells. Lipid production by cells disclosedherein can occur during the log phase or thereafter, including thestationary phase wherein nutrients are supplied, or still available, toallow the continuation of lipid production in the absence of celldivision.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 20% by weight of lipid,preferably at least about 40% by weight, more preferably at least about50% by weight, and most preferably at least about 60% by weight. Processconditions may be adjusted to increase the yield of lipids suitable fora particular use and/or to reduce production cost. For example, incertain embodiments, a microalgae is cultured in the presence of alimiting concentration of one or more nutrients, such as, for example,nitrogen, phosphorous, or sulfur, while providing an excess of fixedcarbon energy such as glucose. Nitrogen limitation tends to increasemicrobial lipid yield over microbial lipid yield in a culture in whichnitrogen is provided in excess. In particular embodiments, the increasein lipid yield is at least about: 10%, 50%, 100%, 200%, or 500%. Themicrobe may be cultured in the presence of a limiting amount of anutrient for a portion of the total culture period or for the entireperiod. In particular embodiments, the nutrient concentration is cycledbetween a limiting concentration and a non-limiting concentration atleast twice during the total culture period. Lipid content of cells maybe increased by continuing the culture for increased periods of timewhile providing an excess of carbon, but limiting or no nitrogen.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example:biotin, pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and may be introduced into microbes (e.g., microalgae), usingcontructs and techniques such as those described above.

The specific examples of bioreactors, culture conditions, andheterotrophic growth and propagation methods described herein may becombined in any suitable manner to improve efficiencies of microbialgrowth and lipid and/or protein production.

Microalgal biomass with a high percentage of oil/lipid accumulation bydry weight has been generated using different methods of culture, whichare known in the art (see PCT Pub. No. 2008/151149). Microalgal biomassgenerated by the culture methods described herein and useful inaccordance with the present invention comprises at least 2% microalgaloil by dry weight. In some embodiments, the microalgal biomass comprisesat least 10%, at least 25%, at least 50%, at least 55%, or at least 60%microalgal oil by dry weight. In some embodiments, the microalgalbiomass contains from 10-90% microalgal oil, from 25-75%.

The microalgal oil of the biomass described herein, or extracted fromthe biomass for use in the methods and compositions of the presentinvention can comprise glycerolipids with one or more distinct fattyacid ester side chains. Glycerolipids are comprised of a glycerolmolecule esterified to one, two or three fatty acid molecules, which maybe of varying lengths and have varying degrees of saturation. The lengthand saturation characteristics of the fatty acid molecules (and themicroalgal oils) may be manipulated to modify the properties orproportions of the fatty acid molecules in the microalgal oils of thepresent invention via culture conditions or via lipid pathwayengineering, as described in more detail in Section IV, below. Thus,specific blends of algal oil may be prepared either within a singlespecies of algae by mixing together the biomass or algal oil from two ormore species of microalgae, or by blending algal oil of the inventionwith oils from other sources such as soy, rapeseed, canola, palm, palmkernel, coconut, corn, waste vegetable, Chinese tallow, olive,sunflower, cottonseed, chicken fat, beef tallow, porcine tallow,microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice whitegrease, lard, Camelina sativa, mustard seed, cashew nut, oats, lupine,kenaf, calendula, help, coffee, linseed (flax), hazelnut, euphorbia,pumpkin seed, coriander, camellia, sesame, safflower, rice, tung tree,cocoa, copra, opium poppy, castor beans, pecan, jojoba, macadamia,Brazil nuts, avocado, petroleum, or a distillate fraction of any of thepreceding oils.

The oil composition, i.e., the properties and proportions of the fattyacid constituents of the glycerolipids, can also be manipulated bycombining biomass or oil from at least two distinct species ofmicroalgae. In some embodiments, at least two of the distinct species ofmicroalgae have different glycerolipid profiles. The distinct species ofmicroalgae may be cultured together or separately as described herein,preferably under heterotrophic conditions, to generate the respectiveoils. Different species of microalgae can contain different percentagesof distinct fatty acid constituents in the cell's glycerolipids

Generally, Prototheca strains have very little or no fatty acids withthe chain length C8-C14. For example, Prototheca moriformis (UTEX 1435),Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442) andPrototheca zopfii (UTEX 1438) contains no (or undectable amounts) C8fatty acids, between 0-0.01% C10 fatty acids, between 0.03-2.1% C12fatty acids and between 1.0-1.7% C14 fatty acids.

Microalgal oil can also include other constituents produced by themicroalgae, or incorporated into the microalgal oil from the culturemedium. These other constituents may be present in varying amountdepending on the culture conditions used to culture the microalgae, thespecies of microalgae, the extraction method used to recover microalgaloil from the biomass and other factors that may affect microalgal oilcomposition. Non-limiting examples of such constituents includecarotenoids, present from 0.025-0.3 mcg/g, preferably from 0.05 to 0.244micrograms/gram, of oil; chlorophyll A present from 0.025-0.3 mcg/g,preferably from 0.045 to 0.268 micrograms/gram, of oil; totalchlorophyll of less than 0.03 mcg/g, preferably less than 0.025micrograms/gram, of oil; gamma tocopherol present from 35-175 mcg/g,preferably from 38.3-164 micrograms/gram, of oil; total tocopherolspresent from 50-300 mcg/g, preferably from 60.8 to 261.7 microgram/gram,of oil; less than 0.5%, preferably less than 0.25%, brassicasterol,campesterol, stigmasterol, or betasitosterol; total tocotrienols lessthan 300 micrograms/gram of oil; and total tocotrienols present from225-350 mcg/g, preferably from 249.6 to 325.3 micrograms/gram, of oil.

Other constituents can include, without limitation, phospholipids,tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene,beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin,alpha-cryptoxanthin and beta-crytoxanthin), and various organic orinorganic compounds. In some cases, the oil extracted from Protothecaspecies comprises between 0.001 to 0.05, preferably from 0.003 to 0.039,microgram lutein/gram of oil, less than 0.005, preferably less than0.003, micrograms lycopene/gram of oil; and less than 0.005, preferablyless than 0.003, microgram beta carotene/gram of oil.

The stable carbon isotope value δ13C is an expression of the ratio of13C/12C relative to a standard (e.g. PDB, carbonite of fossil skeletonof Belemnite americana from Peedee formation of South Carolina). Thestable carbon isotope value δ13C (‰) of the oils can be related to theδ13C value of the feedstock used. In some embodiments the oils arederived from oleaginous organisms heterotrophically grown on sugarderived from a C4 plant such as corn or sugarcane. In some embodimentsthe δ13C (‰) of the oil is from −10 to −17‰ or from −13 to −16‰.

In a second aspect, the present invention provides methods for obtainingmicrobial oil comprising culturing a genetically engineered Protothecacell of the invention under conditions such that oil is produced. Invarious embodiments, the microbial oil has a fatty acid profile selectedfrom the group consisting of: 0.1% to 60% C8:0, 0.1% to 60% C10:0, 0.1%to 60% C12:0, 0.1% to 95% C14:0, 1% to 95% C16:0, 0.1% to 95% C18:0,0.1% to 95% C18:1, 0.1% to 60% C18:2, 0.1% to 60% C18:3 or combinationsthereof. In various embodiments, the ratio of C10:0 to C12:0 is at least3:1. In some cases, the ratio of C10:0 to C14:0 is at least 10:1. Insome cases, the ratio of C10:0 to C14:0 is at least 2:1. In some cases,the ratio of C12:0 to C14:0 is at least 2:1. In some cases, the ratio ofC12:0 to C14:0 is at least 3:1. In various embodiments, the geneticallyengineered cell has a fatty acid profile of at least 40% saturated fattyacids, of at least 60% saturated fatty acids, or at least 85% saturatedfatty acids. In various embodiments, the genetically engineered cell hasa fatty acid profile of at least 85% unsaturated fatty acids, of atleast 90% unsaturated fatty acids, of at least 95% unsaturated fattyacids, or at least 97% unsaturated fatty acids.

In a third aspect, the present invention provides microbial oils andfoods, fuels, and chemicals containing said oil or a chemical derivedtherefrom.

Section VI. Nucleic Acids

In a fifth aspect, the present invention provides recombinant nucleicacids useful in methods for making genetically modified Prototheca andother cells. Embodiments of the present invention providepolynucleotides that encode some portion of a coding sequence of aPrototheca lipid biosynthesis gene.

In various embodiments, these nucleic acids include expressioncassettes, which consist of a coding sequence and control sequences thatregulate expression of the coding sequence, which may code for an mRNAthat encodes a lipid biosynthesis enzyme or for an RNAi that acts tosuppress expression of a fatty acid biosynthesis gene.

In other embodiments, these nucleic acids are expression vectors thatinclude one or more expression cassettes and stably replicate in aPrototheca or other host cell, either by integration into chromosomalDNA of the host cell or as freely replicating plasmids.

In other embodiments, these nucleic acids comprise a portion of aPrototheca lipid biosynthesis gene, which portion may be a portion of acoding sequence, an exon, or a control element. Such nucleic acids areuseful in the construction of expression cassettes for Prototheca andnon-Prototheca host cells, for integration of exogenous DNA intoPrototheca host cells, for regulating expression of exogenous genesexpressed in Prototheca and non-Prototheca host cells, and forconstruction of nucleic acids useful for inactivating Prototheca lipidsynthesis genes such as through homologous recombination or antisenseRNA mediated knockdown.

EXAMPLES Example 1 Methods for Culturing Prototheca

Prototheca strains were cultivated to achieve a high percentage of oilby dry cell weight. Cryopreserved cells were thawed at room temperatureand 500 ul of cells were added to 4.5 ml of medium (4.2 g/L K₂HPO₄, 3.1g/L NaH₂PO₄, 0.24 g/L MgSO₄.7H₂O, 0.25 g/L Citric Acid monohydrate,0.025 g/L CaCl₂ 2H₂O, 2 g/L yeast extract) plus 2% glucose and grown for7 days at 28° C. with agitation (200 rpm) in a 6-well plate. Dry cellweights were determined by centrifuging 1 ml of culture at 14,000 rpmfor 5 min in a pre-weighed Eppendorf tube. The culture supernatant wasdiscarded and the resulting cell pellet washed with 1 ml of deionizedwater. The culture was again centrifuged, the supernatant discarded, andthe cell pellets placed at −80° C. until frozen. Samples were thenlyophilized for 24 hrs and dry cell weights calculated. Fordetermination of total lipid in cultures, 3 ml of culture was removedand subjected to analysis using an Ankom system (Ankom Inc., Macedon,N.Y.) according to the manufacturer's protocol. Samples were subjectedto solvent extraction with an Amkom XT10 extractor according to themanufacturer's protocol. Total lipid was determined as the difference inmass between acid hydrolyzed dried samples and solvent extracted, driedsamples. Percent oil dry cell weight measurements are shown in Table 3.

TABLE 3 Percent oil by dry cell weight Species Strain % Oil Protothecastagnora UTEX 327 13.14 Prototheca moriformis UTEX 1441 18.02 Protothecamoriformis UTEX 1435 27.17

Microalgae samples from multiple strains from the genus Prototheca weregenotyped. Genomic DNA was isolated from algal biomass as follows. Cells(approximately 200 mg) were centifuged from liquid cultures 5 minutes at14,000×g. Cells were then resuspended in sterile distilled water,centrifuged 5 minutes at 14,000×g and the supernatant discarded. Asingle glass bead ˜2 mm in diameter was added to the biomass and tubeswere placed at −80° C. for at least 15 minutes. Samples were removed and150 μl of grinding buffer (1% Sarkosyl, 0.25 M Sucrose, 50 mM NaCl, 20mM EDTA, 100 mM Tris-HCl, pH 8.0, RNase A 0.5 ug/ul) was added. Pelletswere resuspended by vortexing briefly, followed by the addition of 40 ulof 5M NaCl. Samples were vortexed briefly, followed by the addition of66 μl of 5% CTAB (Cetyl trimethylammonium bromide) and a final briefvortex. Samples were next incubated at 65° C. for 10 minutes after whichthey were centrifuged at 14,000×g for 10 minutes. The supernatant wastransferred to a fresh tube and extracted once with 300 μl ofPhenol:Chloroform:Isoamyl alcohol 12:12:1, followed by centrifugationfor 5 minutes at 14,000×g. The resulting aqueous phase was transferredto a fresh tube containing 0.7 vol of isopropanol (˜190 μl), mixed byinversion and incubated at room temperature for 30 minutes or overnightat 4° C. DNA was recovered via centrifugation at 14,000×g for 10minutes. The resulting pellet was then washed twice with 70% ethanol,followed by a final wash with 100% ethanol. Pellets were air dried for20-30 minutes at room temperature followed by resuspension in 50 μl of10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total algal DNA, prepared as described above, was diluted1:50 in 10 mM Tris, pH 8.0. PCR reactions, final volume 20 μl, were setup as follows. Ten μl of 2× iProof HF master mix (BIO-RAD) was added to0.4 μl primer SZ02613 (5′-TGTTGAAGAATGAGCCGGCGAC-3′) at 10 mM stockconcentration. This primer sequence runs from position 567-588 inGenBank accession no. L43357 and is highly conserved in higher plantsand algal plastid genomes. This was followed by the addition of 0.4 μlprimer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′) at 10 mM stockconcentration. This primer sequence is complementary to position1112-1093 in GenBank accession no. L43357 and is highly conserved inhigher plants and algal plastid genomes. Next, 5 μl of diluted total DNAand 3.2 μl dH₂O were added. PCR reactions were run as follows: 98° C.,45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cycles followed by 72°C. for 1 min and holding at 25° C. For purification of PCR products, 20μl of 10 mM Tris, pH 8.0, was added to each reaction, followed byextraction with 40 ρl of Phenol:Chloroform:isoamyl alcohol 12:12:1,vortexing and centrifuging at 14,000×g for 5 minutes. PCR reactions wereapplied to S-400 columns (GE Healthcare) and centrifuged for 2 minutesat 3,000×g. Purified PCR products were subsequently TOPO cloned intoPCR8/GW/TOPO and positive clones selected for on LB/Spec plates.Purified plasmid DNA was sequenced in both directions using M13 forwardand reverse primers. In total, twelve Prototheca strains were selectedto have their 23S rRNA DNA sequenced and the sequences are listed in theSequence Listing. A summary of the strains and Sequence Listing Numbersis included below. The sequences were analyzed for overall divergencefrom the UTEX 1435 (SEQ ID NO: 5) sequence. Two pairs emerged (UTEX329/UTEX 1533 and UTEX 329/UTEX 1440) as the most divergent. In bothcases, pairwise alignment resulted in 75.0% pairwise sequence identity.The percent sequence identity to UTEX 1435 is also included below:

Species Strain % nt identity SEQ ID NO. Prototheca kruegani UTEX 32975.2 SEQ ID NO: 1 Prototheca wickerhamii UTEX 1440 99 SEQ ID NO: 2Prototheca stagnora UTEX 1442 75.7 SEQ ID NO: 3 Prototheca moriformisUTEX 288 75.4 SEQ ID NO: 4 Prototheca moriformis UTEX 100 SEQ ID NO: 51439; 1441; 1435; 1437 Prototheca wikerhamii UTEX 1533 99.8 SEQ ID NO: 6Prototheca moriformis UTEX 1434 75.9 SEQ ID NO: 7 Prototheca zopfii UTEX1438 75.7 SEQ ID NO: 8 Prototheca moriformis UTEX 1436 88.9 SEQ ID NO: 9

Lipid samples from a subset of the above-listed strains were analyzedfor fatty acid profile using HPLC. Results are shown below in Table 4.

TABLE 4 Diversity of fatty acid chains in Prototheca species StrainC14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 UTEX 0 12.01 0 050.33 17.14 0 0 0  327 UTEX 1.41 29.44 0.70 3.05 57.72 12.37 0.97 0.33 01441 UTEX 1.09 25.77 0 2.75 54.01 11.90 2.44 0 0 1435

Oil extracted from Prototheca moriformis UTEX 1435 (via solventextraction or using an expeller press) was analyzed for carotenoids,chlorophyll, tocopherols, other sterols and tocotrienols. The resultsare summarized below in Table 5.

TABLE 5 Carotenoid, chlorophyll, tocopherol/sterols and tocotrienolanalysis in oil extracted from Prototheca moriformis (UTEX 1435).Pressed oil Solvent extracted (mcg/ml) oil (mcg/ml) cis-Lutein 0.0410.042 trans-Lutein 0.140 0.112 trans-Zeaxanthin 0.045 0.039cis-Zeaxanthin 0.007 0.013 t-alpha-Crytoxanthin 0.007 0.010t-beta-Crytoxanthin 0.009 0.010 t-alpha-Carotene 0.003 0.001c-alpha-Carotene none detected none detected t-beta-Carotene 0.010 0.0099-cis-beta-Carotene 0.004 0.002 Lycopene none detected none detectedTotal Carotenoids 0.267 0.238 Chlorophyll <0.01 mg/kg <0.01 mg/kgTocopherols and Sterols Pressed oil Solvent extracted (mg/100 g) oil(mg/100 g) gamma Tocopherol 0.49 0.49 Campesterol 6.09 6.05 Stigmasterol47.6 47.8 Beta-sitosterol 11.6 11.5 Other sterols 445 446 TocotrienolsPressed oil Solvent extracted (mg/g) oil (mg/g) alpha Tocotrienol 0.260.26 beta Tocotrienol <0.01 <0.01 gamma Tocotrienol 0.10 0.10 detalTocotrienol <0.01 <0.01 Total Tocotrienols 0.36 0.36

Oil extracted from Prototheca moriformis, from four separate lots, wasrefined and bleached using standard vegetable oil processing methods.Briefly, crude oil extracted from Prototheca moriformis was clarified ina horizontal decanter, where the solids were separated from the oil. Theclarified oil was then transferred to a tank with citric acid and waterand left to settle for approximately 24 hours. After 24 hours, themixture in the tank formed 2 separate layers. The bottom layer wascomposed of water and gums that were then removed by decantation priorto transferring the degummed oil into a bleaching tank. The oil was thenheated along with another dose of citric acid. Bleaching clay was thenadded to the bleaching tank and the mixture was further heated undervacuum in order to evaporate off any water that was present. The mixturewas then pumped through a leaf filter in order to remove the bleachingclay. The filtered oil was then passed through a final 5 μm polishingfilter and then collected for storage until use. The refined andbleached (RB) oil was then analyzed for carotenoids, chlorophyll,sterols, tocotrienols and tocopherols. The results of these analyses aresummarized in Table 6 below. “Nd” denotes none detected and thesensitivity of detection is listed below:

Sensitivity of Detection

Carotenoids (mcg/g) nd=<0.003 mcg/g

Chlorophyll (mcg/g) nd=<0.03 mcg/g

Sterols (%) nd=0.25%

Tocopherols (mcg/g); nd=3 mcg/g

TABLE 6 Carotenoid, chlorophyll, sterols, tocotrienols and tocopherolanalysis from refined and bleached Prototheca moriformis oil. Lot A LotB Lot C Lot D Carotenoids (mcg/g) Lutein 0.025 0.003 nd 0.039 Zeaxanthinnd nd nd nd cis-Lutein/Zeaxanthin nd nd nd nd trans-alpha-Cryptoxanthinnd nd nd nd trans-beta-Cryptoxanthin nd nd nd nd trans-alpha-Carotene ndnd nd nd cis-alpha-Carotene nd nd nd nd trans-beta-Carotene nd nd nd ndcis-beta-Carotene nd nd nd nd Lycopene nd nd nd nd Unidentified 0.2190.066 0.050 0.026 Total Carotenoids 0.244 0.069 0.050 0.065 Chlorophyll(mcg/g) Chlorophyll A 0.268 0.136 0.045 0.166 Chlorophyll B nd nd nd ndTotal Chlorophyll 0.268 0.136 0.045 0.166 Sterols (%) Brassicasterol ndnd nd nd Campesterol nd nd nd nd Stigmasterol nd nd nd ndbeta-Sitosterol nd nd nd nd Total Sterols nd nd nd nd Tocopherols(mcg/g) alpha-Tocopherol 23.9 22.8 12.5 8.2 beta-Tocopherol 3.72 nd ndnd gamma-Tocopherol 164 85.3 43.1 38.3 delta-Tocopherol 70.1 31.1 18.114.3 Total Tocopherols 262 139.2 73.7 60.8 Tocotrienols (mcg/g)alpha-Tocotrienol 190 225 253 239 beta-Tocotrienol nd nd nd ndgamma-Tocotrienol 47.3 60.4 54.8 60.9 delta-Tocotrienol 12.3 16.1 17.515.2 Total Tocotrienols 250 302 325 315

The same four lots of Prototheca moriformis oil was also analyzed fortrace elements and the results are summarized below in Table 7.

TABLE 7 Elemental analysis of refined and bleached Prototheca moriformisoil. Lot A Lot B Lot C Lot D Elemental Analysis (ppm) Calcium 0.08 0.07<0.04 0.07 Phosphorous <0.2 0.38 <0.2 0.33 Sodium <0.5 0.55 <0.5 <0.5Potassium 1.02 1.68 <0.5 0.94 Magnesium <0.04 <0.04 <0.04 0.07 Manganese<0.05 <0.05 <0.05 <0.05 Iron <0.02 <0.02 <0.02 <0.02 Zinc <0.02 <0.02<0.02 <0.02 Copper <0.05 <0.05 <0.05 <0.05 Sulfur 2.55 4.45 2.36 4.55Lead <0.2 <0.2 <0.2 <0.2 Silicon 0.37 0.41 0.26 0.26 Nickel <0.2 <0.2<0.2 <0.2 Organic chloride <1.0 <1.0 <1.0 2.2 Inorganic chloride <1.0<1.0 <1.0 <1.0 Nitrogen 4.4 7.8 4.2 6.9 Lithium <0.02 <0.02 <0.02 <0.02Boron 0.07 0.36 0.09 0.38 Aluminum — <0.2 <0.2 <0.2 Vanadium <0.05 <0.05<0.05 <0.05 Lovibond Color (°L) Red 5.0 4.3 3.2 5.0 Yellow 70.0 70.050.0 70.0 Mono & Diglycerides by HPLC (%) Diglycerides 1.68 2.23 1.251.61 Monoglycerides 0.03 0.04 0.02 0.03 Free fatty acids (FFA) 1.02 1.720.86 0.83 Soaps 0 0 0 Oxidized and Polymerized Triglycerides OxidizedTriglycerides 3.41 2.41 4.11 1.00 (%) Polymerized 1.19 0.45 0.66 0.31Triglycerides (%) Peroxide Value 0.75 0.80 0.60 1.20 (meg/kg)p-Anisidine value 5.03 9.03 5.44 20.1 (dimensionless) Water and OtherImpurities (%) Karl Fisher Moisture 0.8 0.12 0.07 0.18 Total polar 5.026.28 4.54 5.23 compounds Unsaponificable 0.92 1.07 0.72 1.04 matterInsoluble impurities <0.01 <0.01 0.01 <0.01 Total oil (%) Neutral oil98.8 98.2 99.0 98.9

Example 2 General Methods for Biolistic Transformation of Prototheca

Seashell Gold Microcarriers (550 nanometers) were prepared according tothe protocol from manufacturer. Plasmid (20 μg) was mixed with 50 μl ofbinding buffer and 60 μl (30 mg) of S550d gold carriers and incubated inice for 1 min. Precipitation buffer (100 μl) was added, and the mixturewas incubated in ice for another 1 min. After vortexing, DNA-coatedparticles were pelleted by spinning at 10,000 rpm in an Eppendorf 5415Cmicrofuge for 10 seconds. The gold pellet was washed once with 500 μl ofcold 100% ethanol, pelleted by brief spinning in the microfuge, andresuspended with 50 μl of ice-cold ethanol. After a brief (1-2 sec)sonication, 10 μl of DNA-coated particles were immediately transferredto the carrier membrane.

Prototheca strains were grown in proteose medium (2 g/L yeast extract,2.94 mM NaNO3, 0.17 mM CaCl2.2H2O, 0.3 mM MgSO4.7H2O, 0.4 mM K2HPO4,1.28 mM KH2PO4, 0.43 mM NaCl) with 2% glucose on a gyratory shaker untilit reaches a cell density of 2×10⁶cells/ml. The cells were harvested,washed once with sterile distilled water, and resuspended in 50 μl ofmedium. 1×10⁷ cells were spread in the center third of a non-selectiveproteose media plate. The cells were bombarded with the PDS-1000/HeBiolistic Particle Delivery system (Bio-Rad). Rupture disks (1350 psi)were used, and the plates are placed 6 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 h. Uponrecovery, the cells were scraped from the plates with a rubber spatula,mixed with 100 μl of medium and spread on plates containing theappropriate antibiotic selection. After 7-10 days of incubation at 25°C., colonies representing transformed cells were visible on the plates.Colonies were picked and spotted on selective (either antibiotic orcarbon source) agar plates for a second round of selection.

Example 3 Fatty Acid Analysis by Fatty Acid Methyl Ester Detection

Lipid samples were prepared from dried biomass. 20-40 mg of driedbiomass was resuspended in 2 mL of 5% H₂SO₄ in MeOH, and 200 ul oftoluene containing an appropriate amount of a suitable internal standard(C19:0) was added. The mixture was sonicated briefly to disperse thebiomass, then heated at 70-75° C. for 3.5 hours. 2 mL of heptane wasadded to extract the fatty acid methyl esters, followed by addition of 2mL of 6% K₂CO₃ (aq) to neutralize the acid. The mixture was agitatedvigorously, and a portion of the upper layer was transferred to a vialcontaining Na₂SO₄ (anhydrous) for gas chromatography analysis usingstandard FAME

Example 4 Altering the Levels of Saturated Fatty Acids in the MicroalgaePrototheca Moriformis A. Decreasing Stearoyl ACP Desaturase and Delta 12Fatty Acid Desaturase Expression by a Gene Knockout Approach

As part of a genomics screen using a bioinformatics based approach basedon cDNAs, Illumina transcriptome and Roche 454 sequencing of genomic DNAfrom Prototheca moriformis (UTEX 1435), two specific groups of genesinvolved in fatty acid desaturation were identified: stearoyl ACPdesaturases (SAD) and delta 12 fatty acid desaturases (Δ12 FAD).Stearoyl ACP desaturase enzymes are part of the lipid synthesis pathwayand they function to introduce double bonds into the fatty acyl chains,for example, the synthesis of C18:1 fatty acids from C18:0 fatty acids.Delta 12 fatty acid desaturases are also part of the lipid synthesispathway and they function to introduce double bonds into alreadyunsaturated fatty acids, for example, the synthesis of C18:2 fatty acidsfrom C18:1 fatty acids. The genes encoding stearoyl ACP desaturases fellinto two distinct families. Based on these results, three genedisruption constructs were designed to potentially disrupt multiple genefamily members by targeting more highly conserved coding regions withineach family of desaturase enzymes.

Three homologous recombination targeting constructs were designed using:(1) highly conserved portions of the coding sequence of delta 12 fattyacid desaturase (d12FAD) or FAD2 family members and (2) two constructstargeting each of the two distinct families of SAD (SAD1 and SAD2), eachwith conserved regions of the coding sequences from each family. Thisstrategy would embed a selectable marker gene into these highlyconserved coding regions (targeting multiple family members) rather thana classic gene replacement strategy where the homologous recombinationwould target flanking regions of the targeted gene.

All constructs were introduced into the cells by biolistictransformation using the methods described above and constructs werelinearized before being shot into the cells. Transformants were selectedon sucrose containing plates/media and changes in lipid profile wereassayed using the above-described method. Relevant sequences from eachof the three targeting constructs are listed below.

Description SEQ ID NO: 5′ sequence from coding region of d12FAD SEQ IDNO: 18 targeting construct 3′ sequence from coding region of d12FAD SEQID NO: 19 targeting construct d12FAD targeting construct cDNA sequenceSEQ ID NO: 20 5′ sequence from coding region of SAD2A SEQ ID NO: 21 3′sequence from coding region of SAD2A SEQ ID NO: 22 SAD2A targetingconstruct cDNA sequence SEQ ID NO: 23 5′ sequence from coding region osSAD2B SEQ ID NO: 24 3′ sequence from coding region of SAD2B SEQ ID NO:25 SAD2B targeting construct cDNA sequence SEQ ID NO: 26

Representative positive clones from transformations with each of theconstructs were picked and the fatty acid profiles for these clones weredetermined. Lipid samples were prepared from dried biomass from eachtransformant and fatty acid profiles from these samples were analyzedusing standard fatty acid methyl ester gas chromatography flameionization (FAME GC/FID) detection methods as described in Example 3.The fatty acid profiles (expressed as Area % of total fatty acids) fromthe transgenic lines arising from transformation are shown in Table 8.

TABLE 8 Fatty acid profiles of desaturase knockouts. Fatty Acid d12FADKO SAD2A KO SAD2B KO wt UTEX 1435 C8:0 0 0 0 0 C10:0 0.01 0.01 0.01 0.01C12:0 0.03 0.03 0.03 0.03 C14:0 1.08 0.985 0.795 1.46 C16:0 24.42 25.33523.66 29.87 C18:0 6.85 12.89 19.555 3.345 C18:1 58.35 47.865 43.11554.09 C18:2 7.33 10.27 9.83 9.1 C18:3 alpha 0.83 0.86 1 0.89 C20:0 0.480.86 1.175 0.325

Each construct had a measurable impact on the desired class of fattyacid and in all three cases C18:0 levels increased markedly,particularly with the two SAD knockouts. Further comparison of multipleclones from the SAD knockouts indicated that the SAD2B knockout lineshad significantly greater reductions in C18:1 fatty acids than the C18:1fatty acid levels observed with the SAD2A knockout lines.

Additional Δ12 FAD knockouts were generated in a Prototheca moriformisbackground using the methods described above. In order to identifypotential homologous of Δ12FADs, the following primers were used inorder to amplify a genomic region encoding a putative FAD:

Primer 1  SEQ ID NO: 27 5′-TCACTTCATGCCGGCGGTCC-3′ Primer 2 SEQ ID NO: 28 5′-GCGCTCCTGCTTGGCTCGAA-3′The sequences resulting from the genomic amplification of Protothecamoriformis genomic DNA using the above primers were highly similar, butindicated that multiple genes or alleles of Δ12FADs exist in Prototheca.

Based on this result, two gene disruption constructs were designed thatsought to inactivate one or more Δ12FAD genes. The strategy would embeda sucrose invertase (suc2 from S. cerevisiae) cassette (SEQ ID NO: 29),thus conferring the ability to hydrolyze sucrose as a selectable marker,into highly conserved coding regions rather than use a classic genereplacement strategy. The first construct, termed pSZ1124, contained 5′and 3′ genomic targeting sequences flanking a C. reinhardtii β-tubulinpromoter (SEQ ID NO: 30) driving the expression of the S. cerevisiaesuc2 gene (SEQ ID NO: 31) and a Chlorella vulgaris nitrate reductase3′UTR (SEQ ID NO: 32). The second construct, termed pSZ1125, contained5′ and 3′ genomic targeting sequences flanking a C. reinhardtiiβ-tubulin promoter driving the expression of the S. cerevisiae suc2 geneand a Chlorella vulgaris nitrate reductase 3′UTR. The relevant sequencesof the constructs are listed in the Sequence Listing:

S. cerevisiae suc2 cassette SEQ ID NO: 29 pSZ1124 (FAD2B) 5′ genomictargeting sequence SEQ ID NO: 33 pSZ1124 (FAD2B) 3′ genomic targetingsequence SEQ ID NO: 34 pSZ1125 (FAD2C) 5′ genomic targeting sequence SEQID NO: 35 pSZ1125 (FAD2C) 3′ genomic targeting sequence SEQ ID NO: 36

pSZ1124 and pSZ1125 were each introduced into a Prototheca moriformisbackground. Positive clones were selected based on the ability tohydrolyze sucrose. Table 9 summarizes the fatty acid profiles (in Area%, generated using methods described above) obtained in two transgeniclines in which pSZ1124 and pSZ1125 targeting vectors were utilized.

TABLE 9 Fatty acid profiles of Δ12 FAD knockouts C10:0 C12:0 C14:0 C16:0C16:1 C18:0 C18:1 C18:2 C18:3α parent 0.01 0.03 1.15 26.13 1.32 4.3957.20 8.13 0.61 FAD2B 0.02 0.03 0.80 12.84 1.92 0.86 74.74 7.08 0.33FAD2C 0.02 0.04 1.42 25.85 1.65 2.44 66.11 1.39 0.22

The transgenic containing the FAD2B (pSZ1124) construct gave a veryinteresting and unexpected result in fatty acid profile, in that theC18:2 levels, which would be expected to decrease, only decreased byabout one area %. However, the C18:1 fatty acid levels increasedsignificantly, almost exclusively at the expense of the C16:0 levels,which decreased significantly. The transgenic containing the FAD2C(pSZ1125) construct also gave a change in lipid profile: the levels ofC18:2 are reduced significantly along with a corresponding increase inC18:1 levels.

B. RNA Hairpin Approach to Down-Regulation of Delta 12 Desaturase (FADc)in Prototheca Cells

Vectors constructed to down-regulate FADc (delta 12 desaturase gene)gene expression by long hairpin RNAs were introduced into a Protothecamoriformis UTEX 1435 genetic background. The Saccharomyces cerevisiaesuc2 sucrose invertase gene was utilized as a selectable marker,conferring the ability to grow on sucrose as a sole-carbon source topositive clones, and two types of constructs were used. The first typeof construct utilized a portion of the first exon of the FADc codingregion linked in cis to its first intron followed by a repeat unit ofthe first exon in reverse orientation. This type of construct wasdesigned to form a hairpin when expressed as mRNA. Two constructs ofthis first type were created, one driven by the Prototheca moriformisAmt03 promoter (SEQ ID NO:37), termed pSZ1468, and a second driven bythe Chlamydomomas reinhardtii β-tubulin promoter (SEQ ID NO:30), termedpSZ1469. The second type of construct utilized the large FADc exon 2 inthe antisense orientation driven by either the Prototheca moriformisAmt03 promoter (SEQ ID NO:37), termed pSZ1470, or driven by theChlamydomomas reinhardtii β-tubulin promoter (SEQ ID NO:30), termedpSZ1471. All four constructs had a S. cerevisiae suc2 sucrose invertasecassette (SEQ ID NO:29) and a 5′ (SEQ ID NO:38) and 3′ (SEQ ID NO:39)homologous recombination targeting sequences (flanking the construct) tothe 6S genomic region for integration into the nuclear genome. Sequencesof the FADc portions of each long hairpin RNA construct along with therelevant portions of each construct are listed in the Sequence Listingas:

Description SEQ ID NO: pSZ1468 FADc RNA hairpin cassette SEQ ID NO: 40Relevant portions of the pSZ1468 construct SEQ ID NO: 41 pSZ1469 FADcRNA hairpin cassette SEQ ID NO: 42 Relevant portions of the pSZ1469construct SEQ ID NO: 43 pSZ1470 FADc exon 2 RNA hairpin cassette SEQ IDNO: 44 Relevant portions of the pSZ1470 construct SEQ ID NO: 45 pSZ1471FADc exon 2 RNA hairpin cassette SEQ ID NO: 46 Relevant portions of thepSZ1471 construct SEQ ID NO: 47

Each of the four constructs was transformed into a Prototheca moriformis(UTEX 1435) background and positive clones were screened using plateswith sucrose as the sole carbon source. Positive clones were picked fromeach transformation and a subset were selected to determine the impactof the hairpin and antisense cassettes contained in pSZ1468, pSZ1469,pSZ1470 and pSZ1471 on fatty acid profiles. The selected clones fromeach transformation were grown under lipid producing conditions and thefatty acid profiles were determined using direct transesterificationmethods as described above. Representative fatty acid profiles from eachof the transformations are summarized below in Table 10. Wildtype 1 and2 cells were untransformed Prototheca moriformis (UTEX 1435) cells thatwere run with each of the transformants as a negative control.

TABLE 10 Fatty acid profiles of Prototheca moriformis cells containinglong hairpin RNA constructs to down-regulate the expression of delta 12desaturase gene (FADc). Strain C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2wildtype 1 0.01 0.03 1.20 27.08 4.01 57.58 7.81 pSZ1468 0.01 0.04 1.3325.95 3.68 65.60 1.25 clone A pSZ1468 0.01 0.03 1.18 23.43 2.84 65.324.91 clone B pSZ1468 0.01 0.04 1.34 23.18 4.27 63.65 5.17 clone CpSZ1468 0.01 0.03 1.24 23.00 3.85 61.92 7.62 clone D pSZ1470 0.01 0.031.23 24.79 4.33 58.43 8.92 clone A pSZ1470 0.01 0.03 1.26 24.91 4.1457.59 9.64 clone B pSZ1470 0.01 0.03 1.21 23.35 4.75 58.52 9.70 clone Cwildtype 2 0.01 0.03 0.98 24.65 3.68 62.48 6.26 pSZ1469 0.01 0.03 1.0521.74 2.71 71.33 1.22 clone A pSZ1469 0.01 0.03 1.01 22.60 2.98 70.191.27 clone B pSZ1469 0.01 0.03 1.03 19.82 2.38 72.95 1.82 clone CpSZ1469 0.01 0.03 1.03 20.54 2.66 70.96 2.71 clone D pSZ1471 0.01 0.031.03 18.42 2.63 66.94 8.55 clone A pSZ1471 0.01 0.03 0.94 18.61 2.5867.13 8.66 clone B pSZ1471 0.01 0.03 1.00 18.31 2.46 67.41 8.71 clone CpSZ1471 0.01 0.03 0.93 18.82 2.54 66.84 8.77 clone D

The above results show that the hairpin constructs pSZ1468 and pSZ1469showed expected phenotypes: a reduction in C18:2 fatty acid levels andan increase in C18:1 fatty acid levels as compared to wildtype 1 andwildtype 2, respectively. The antisense constructs, pSZ1470 and pSZ1471did not result in a decrease in C18:2 fatty acid levels but insteadshowed a slight increase when compared to wildtype 1 and wildtype 2,respectively and a slight decrease in C16:0 fatty acid levels.

Example 5 Engineered Microalgae with Altered Fatty Acid Profiles

As described above, integration of heterologous genes to attenuatespecific endogenous lipid pathway genes, through knockout or knockdown,in Prototheca species can alter fatty acid profiles. Plasmid constructs(listed in Table 11) were created to assess whether the fatty acidprofile of a host cell may be affected as a result of a knockout anendogenous fatty acyl-ACP thioesterase gene, FATA1.

A classically mutagenized derivative of Protheca moriformis UTEX 1435,Strain J, was transformed with one of the following plasmid constructsin Table 11 using the methods of Example 2. Each construct contained aregion for integration into the nuclear genome to interrupt theendogenous FATA1 gene and a S. cerevisiae suc2 sucrose invertase codingregion under the control of C. reinhardtii β-tubulin promoter/5′UTR (SEQID NO: 30) and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO:32). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO:29 and served as a selection marker. All protein coding regions werecodon optimized to reflect the codon bias inherent in Protothecamoriformis UTEX 1435 (see Table 2) nuclear genes. Relevant sequences forthe targeting regions for the FATA1 gene used for nuclear genomeintegration are shown below.

Description SEQ ID NO: 5′ sequence for integration into FATA1 locus SEQID NO: 48 3′ sequence for integration into FATA1 locus SEQ ID NO: 49

TABLE 11 Plasmid constructs used to transform Protheca moriformis (UTEX1435) STRAIN J. Plasmid Construct Relevant Sequence Elements SEQ ID NO:pSZ1883 FATA1-CrbTub_yInv_nr-FATA1 SEQ ID NO: 50 pSZ1925 FATA1- SEQ IDNO: 51 CrbTub_yInv_nr::amt03_CwTE2_nr- FATA1

To introduce the Cuphea wrightii ACP-thioesterase 2 (CwFatB2) gene(Accession No: U56104) into STRAIN J at the FATA1-1 locus, a constructwas generated to express the protein coding region of the CwFatB2 geneunder the control of the Prototheca moriformis Amt03 promoter/5′UTR (SEQID NO: 37) and C. vulgaris nitrate reductase 3′UTR (SEQ ID NO: 32).Relevant portions of this construct are provided in the Sequence Listingas SEQ ID NO: 51. The codon-optimized cDNA sequences and amino acidsequences of the Cuphea wrightii FatB2 thioesterase are listed in theSequence Listing as SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

Upon transformation of FATA1-CrbTub_yInv_nr-FATA1 into STRAIN J, primarytransformants were clonally purified and grown under standard lipidproduction conditions at pH 5.0 similar to the conditions as disclosedin Example 1. Lipid samples were prepared from dried biomass from eachtransformant and fatty acid profiles from these samples were analyzedusing standard fatty acid methyl ester gas chromatography flameionization (FAME GC/FID) detection methods as described in Example 3.The fatty acid profiles (expressed as Area % of total fatty acids) fromthe transgenic lines arising from transformation with pSZ1883 intoStrain J are shown in Table 12.

TABLE 12 Fatty acid profiles of Prototheca moriformis cells containing aselectable marker to disrupt an endogenous FATA1 allele. % % % % %Transformation C14:0 C16:0 C18:0 C18:1 C18:2 Wildtype 1.23 25.68 2.8360.54 7.52 Transformant 1 0.86 16.95 1.75 68.44 9.78 Transformant 2 0.8517.33 1.71 68.57 9.31 Transformant 3 0.82 17.40 1.78 68.55 9.22Transformant 4 0.84 17.43 1.78 68.25 9.53 Transformant 5 0.75 17.64 2.0269.02 8.61

These results show that ablation of the host's endogenous FATA1-1 allelealters the fatty acid profile of the engineered microalgae. The impactof targeting a selectable marker to the endogenous FATA1 allele is aclear diminution of C16:0 fatty acid production with an increase inC18:1 fatty acid production.

Upon transformation of FATA1-CrbTub_yInv_nr::amt03_CwTE2_nr-FATA1 intoSTRAIN J, primary transformants were clonally purified and grown understandard lipid production conditions at pH 7.0 with different carbonsources provided to a total concentration of 40 g/L. The sucroseconcentration was 40 g/L. Where only glucose was used as the carbonsource, glucose was provided at 40 g/L. Where glucose and fructose wasused as the carbon source, gluces was provided at 20 g/L and fructosewas provided at 20 g/L. Lipid samples were prepared from dried biomassfrom each transformant and fatty acid profiles from these samples wereanalyzed using standard fatty acid methyl ester gas chromatography flameionization (FAME GC/FID) detection methods as described in Example 3.The fatty acid profiles (expressed as Area % of total fatty acids) fromthe transgenic line arising from transformation with pSZ1925into StrainJ are shown in Table 13. The resulting fatty acid profiles are listed inTable 13.

TABLE 13 Fatty acid profiles of Prototheca moriformis cells containing aselectable marker and an exogenous thioesterase to disrupt an endogenousFATA1 allele. Carbon % % % % % % Transformant source % C10:0 C12:0 C14:0C16:0 C18:0 C18:1 C18:2 Strain J Wildtype Glucose 0.01 0.04 1.38 28.833.00 56.05 8.21 Wildtype Glucose 0.01 0.04 1.50 29.38 3.00 55.29 8.23Wildtype Glucose/ 0.01 0.05 1.48 28.58 3.20 57.14 7.27 Fructose WildtypeGlucose/ 0.01 0.04 1.54 29.05 3.23 56.47 7.32 Fructose >2 1 Glucose/4.29 19.98 9.17 20.68 3.47 34.38 6.37 copies Fructose 2 Glucose/ 3.1116.17 9.91 15.97 1.57 45.72 5.81 Fructose 3 Sucrose 4.84 24.22 11.5619.48 2.67 29.56 6.02 4 Sucrose 3.24 16.67 10.39 16.34 1.43 44.41 6.001-2 1 Glucose/ 0.18 1.64 1.85 14.43 2.12 70.30 7.63 copies Fructose 2Glucose/ 0.18 1.56 1.74 13.56 2.25 71.04 7.72 Fructose 3 Sucrose 0.191.69 1.89 13.79 3.15 69.97 7.68 4 Sucrose 0.15 1.26 1.49 13.44 2.7371.46 7.77

Concordant with targeting a selectable marker alone to the host'sFATA1-1 allele, integration of a selectable marker concomitant with anexogenous thioesterase alters the fatty acid profile of the engineeredmicroalgae. As above, targeting an exogenous gene to the FATA1-1 alleleresults in a clear diminution of C16:0 fatty acid production. Theadditional expression of the CwTE2 thioesterase at the FATA1-1 locusalso impacts mid chain fatty acids and C18:1 fatty acid production to anextent that is dependent upon the level of exogenous thioesteraseactivity present in the transformants analyzed. Genes bordered by repeatunits such as the C. vulgaris nitrate reductase 3′ UTR in constructssuch as FATA1-CrbTub_yInv_nr::amt03_CwTE2_nr-FATA1, may be amplifiedupon integration in the host genome. There is good concordance betweencopy number of the amplified transgene at the target integration siteand thioesterase levels as revealed either by impacts on fatty acidprofiles or recombinant protein accumulation as assessed by westernblotting.

Transgenic lines in which the CwTE2 gene has undergone amplificationshow a marked increase in mid chain (C10:0-C14:0) fatty acids and aconcurrent decrease in C18:1 fatty acids. In contrast, thosetransformants in which CwTE2 has undergone little or no amplification(likely 1-2 copies) are consistent with lower expression of theexogenous thioesterase, resulting in a slight increase in mid chainfatty acids and a far greater impact on the increase of C18:1 fattyacids.

Collectively, these data show that ablation of the host's endogenousFATA1-1 allele alters the lipid profile of the engineered microalgae.

Example 6 Altering Fatty Acid Profiles of Microalgae ThroughOverexpression of a Prototheca Lipid Biosynthesis Gene

As described above, the β-ketoacyl-ACP synthase II (KASII) catalyzes the2-carbon extension of C16:0-ACP to C18:0-ACP during fatty acidbiosynthesis. It is an important lipid biosynthesis enzyme inestablishing the fatty acid profile of the host organism and is criticalfor stearate and oleate production. Plasmid constructs were created toassess whether the fatty acid profile of a host cell may be affected asa result of expression of a KASII gene. Sources of KASII gene sequenceswere selected from Protheca moriformis UTEX 1435 or from higher plants(Glycine max, Helianthus annus, or Ricinus communis).

A classically mutagenized derivative of Protheca moriformis UTEX 1435,STRAIN J, was transformed individually with one of the following plasmidconstructs in Table 14 using the methods of Example 2. Each constructcontained a region for integration into the nuclear genome at the 6Slocus and a S. cerevisiae suc2 sucrose invertase coding region under thecontrol of C. reinhardtii β-tubulin promoter/5′UTR and Chlorellavulgaris nitrate reductase 3′ UTR. This S. cerevisiae suc2 expressioncassette is listed as SEQ ID NO: 29 and served as a selection marker.For each construct, the KASII coding region was under the control of thePrototheca moriformis Amt03 promoter/5′UTR (SEQ ID NO: 37) and C.vulgaris nitrate reductase 3′UTR (SEQ ID NO: 32). The native transitpeptide of each KASII enzyme was replaced with the Chlorellaprotothecoides stearoyl-ACP desaturase transit peptide (SEQ ID NO: 54).All protein coding regions were codon optimized to reflect the codonbias inherent in Prototheca moriformis UTEX 1435 nuclear genes (seeTable 2).

Relevant sequences for the targeting regions to the 6S locus for nucleargenome integration are shown below.

Description SEQ ID NO: 5′ sequence for integration into 6S locus SEQ IDNO: 38 3′ sequence for integration into 6S locus SEQ ID NO: 39

TABLE 14 Plasmid constructs used to transform Protheca moriformis (UTEX1435) STRAIN J. Source Plasmid of KASII SEQ Construct enzyme SequenceElements ID. NO: pSZ1747 Glycine max 6S::β- SEQ IDtub:suc2:nr::Amt03:S106SAD: NO: 55 GlmKASII:nr::6S pSZ1750 Helianthus6S::β- SEQ ID annuus tub:suc2:nr::Amt03:S106SAD: NO: 56 HaKASII:nr::6SpSZ1754 Ricinus 6S::β- SEQ ID communis tub:suc2:nr::Amt03:S106SAD: NO:57 RcKASII:nr::6S pSZ2041 Protheca 6S::β- SEQ ID moriformistub:suc2:nr::Amt03:S106SAD: NO: 58 PmKASII:nr::6S

The relevant nucleotide sequence of the construct6S::β-tub:suc2:nr::Amt03:S106SAD:PmKASII:nr::6S is provided in thesequence listings as SEQ ID. NO: 58. The codon-optimized sequence ofPmKASII comprising a Chlorella protothecoides S106 stearoyl-ACPdesaturase transit peptide is provided the sequence listings as SEQ ID.NO: 105. SEQ ID NO: 106 provides the protein translation of SEQ ID NO.105.

Upon individual transformation of each plasmid construct into Strain J,positive clones were screened on plates with sucrose as the sole carbonsource. As in the previous examples, primary transformants were clonallypurified and grown under standard lipid production conditions. Here,transformants were cultivated at pH 7 and lipid samples were preparedfrom dried biomass from each transformant as described above. Fatty acidprofiles (expressed as Area %) of several positive transformants ascompared to a wildtype negative control are summarized for each plasmidconstruct in Table 15 below.

TABLE 15 Fatty acid profiles of Prototheca moriformis cells engineeredto overexpress KAS II genes. Plasmid KASII Construct Source Transformant% C14:0 % C16:0 % C18:0 % C18:1 % C18:2 None no over- 1 1.36 28.69 2.9256.36 8.16 expression 2 1.35 28.13 3.57 55.63 8.79 3 1.22 25.74 2.8260.6 7.31 4 1.22 25.74 2.82 60.6 7.31 pSZ1747 Glm 1 2.23 25.34 2.6957.35 9.53 2 2.18 25.46 2.74 57.35 9.46 3 2.18 25.33 2.89 57.34 9.5 42.2 25.69 2.66 57.28 9.43 5 2.17 25.38 3.03 56.99 9.72 pSZ1750 Ha 1 2.4326.82 2.72 55.17 9.87 2 2.44 27.14 2.62 54.89 9.81 3 2.61 26.9 2.6754.43 10.25 4 1.96 30.32 2.87 53.87 8.26 5 2.55 27.64 2.98 53.82 10.07pSZ1754 Rc 1 1.84 24.41 2.89 59.26 9.08 2 1.3 25.04 2.81 58.75 9.65 31.27 25.98 2.76 58.33 9.22 4 1.95 25.34 2.77 58.15 9.22 5 1.3 26.53 2.7557.87 9.09 pSZ2041 Pm 1 1.63 11.93 3.62 70.95 9.64 2 1.85 11.63 3.3469.88 10.93 3 1.84 12.01 3.81 69.56 10.45 4 1.63 14.22 3.72 68.86 9.6 51.67 15.04 3.05 68.63 9.24

The data presented in Table 15 show that none of the higher plant KASIIgenes effected a change in the fatty acid profile of the transformedmicroalgal cells. Additional plasmid constructs expressing KASII genesfrom higher plants driven by promoters other than the Prototheca Amt03promoter also failed to alter fatty acid profiles in transformed cells.In stark contrast, a clear diminution of C16:0 chain lengths with aconcomitant increase in C18:1 length fatty acids was observed uponoverexpression of the Prototheca moriformis KASII gene codon optimizedusing the codon frequency denoted in Table 2. Similar fatty acid profilechanges were observed upon transformation of constructs expressing thePmKASII gene driven by a β-tublin promoter.

These results show that exogenous expression of a Prototheca lipidbiosynthesis gene can alter the fatty acid profile of geneticallyengineered microalgae.

Example 7 Characteristics of Processed Oil Produced from EngineeredMicroorganisms

Methods and effects of transforming Prototheca moriformis (UTEX 1435)with transformation vector pSZ1500 (SEQ ID NO: 137) have been previouslydescribed in PCT Application Nos. PCT/US2011/038463, PCT/US2011/038464,and PCT/US2012/023696.

A classically mutagenized (for higher oil production) derivative ofProtheca moriformis (UTEX 1435), Strain A, was transformed with pSZ1500according to biolistic transformation methods as described in herein andin PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1500 comprised nucleotidesequence of the Carthamus tinctorius oleyl-thioesterase (CtOTE) gene,codon-optimized for expression in P. moriformis UTEX 1435. The pSZ1500expression construct included 5′ (SEQ ID NO: 138) and 3′ (SEQ ID NO:139) homologous recombination targeting sequences (flanking theconstruct) to the FADc genomic region for integration into the nucleargenome and a S. cerevisiae suc2 sucrose invertase coding region underthe control of C. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5)and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126). ThisS. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 127 andserved as a selection marker. The CtOTE coding region was under thecontrol of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 128) andC. vulgaris nitrate reductase 3′UTR, and the native transit peptide wasreplaced with the C. protothecoides stearoyl-ACP desaturase transitpeptide (SEQ ID NO: 129). The protein coding regions of CtOTE and suc2were codon optimized to reflect the codon bias inherent in P. moriformisUTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

Primary pSZ1500 transformants of Strain A were selected on agar platescontaining sucrose as a sole carbon source, clonally purified, and asingle engineered line, Strain D was selected for analysis. Strain D wasgrown as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Hexaneextraction of the oil from the generated biomass was then performedusing standard methods, and the resulting triglyceride oil wasdetermined to be free of residual hexane. Other methods of extraction ofoil from microalgae using an expeller press are described in PCTApplication No. PCT/US2010/031108 and are hereby incorporated byreference.

Different lots of oil extracted from biomass of Strain D were refined,bleached, and deodorized using standard vegetable oil processingprocessing methods. These procedures generated oil samples RBD437,RBD469, RBD501, RBD 502, RBD503, and RBD529, which were subjected toanalytical testing protocols according to methods defined through theAmerican Oil Chemists' Society, the American Society for Testing andMaterials, and the International Organization for Standardization. Theresults of these analyses are summarized below in Tables 16-21.

TABLE 16 Analytical results for oil sample RBD469. Method Number TestDescription Results Units AOCS Ca 3a-46 Insoluble impurities <0.01 %AOCS Ca 5a-40 Free Fatty Acids (Oleic) 0.02 % AOCS Ca 5a-40 Acid Value0.04 mg KOH/g AOCS CA 9f-57 Neutral oil 98.9 % D97 Cloud Point −15 degC. D97 Pour Point −18 deg C. Karl Fischer Moisture 0.01 % AOCS Cc 13d-55Chlorophyll <0.01 ppm (modified) Iodine Value 78.3 g I₂/100 g AOCS Cd8b-90 Peroxide Value 0.31 meq/kg ISO 6885 p-Anisidine Value 0.65 AOCS Cc18-80 Dropping Melting point 6.2 deg C. (Mettler) AOCS Cd 11d-96Tricylglicerides 98.6 % AOCS Cd 11d-96 Monoglyceride <0.01 % AOCS Cd11d-96 Diglicerides 0.68 % AOCS Cd 20-91 Total Polar Compounds 2.62 %IUPAC, 2.507 and Oxidized & Polymerized 17.62 % 2.508 TricylgliceridesAOCS Cc 9b-55 Flash Point 244 deg C. AOCS Cc 9a-48 Smoke Point 232 degC. AOCS Cd 12b-92 Oxidataive Stability Index 31.6 hours Rancimat (110°C.) AOCS Ca 6a-40 Unsaponified Matter 2.28 %

RBD469 oil was analyzed for trace element content, solid fat content,and Lovibond color according to AOCS methods. Results of these analysesare presented below in Table 17, Table 18, and Table 19.

TABLE 17 ICP Elemental Analysis of RBD469 oil. Method Number TestDescription Results in ppm AOCS Ca 20-99 and Phosphorus 1.09 AOCS Ca17-01 Calcium 0.1 (modified) Magnesium 0.04 Iron <0.02 Sulfur 28.8Copper <0.05 Potassium <0.50 Sodium <0.50 Silicon 0.51 Boron 0.06Aluminum <0.20 Lead <0.20 Lithium <0.02 Nickel <0.20 Vanadium <0.05 Zinc<0.02 Arsenic <0.20 Mercury <0.20 Cadmium <0.03 Chromium <0.02 Manganese<0.05 Silver <0.05 Titanium <0.05 Selenium <0.50 UOP779 Chloride organic<1 UOP779 Chloride inorganic 7.24 AOCS Ba 4e-93 Nitrogen 6.7

TABLE 18 Solid Fat Content of RBD469 Oil Method Number Solid Fat ContentResult AOCS Cd 12b-93 Solid Fat Content 10° C. 0.13% AOCS Cd 12b-93Solid Fat Content 15° C. 0.13% AOCS Cd 12b-93 Solid Fat Content 20° C.0.28% AOCS Cd 12b-93 Solid Fat Content 25° C. 0.14% AOCS Cd 12b-93 SolidFat Content 30° C. 0.08% AOCS Cd 12b-93 Solid Fat Content 35° C. 0.25%

TABLE 19 Lovibond Color of RBD469 Oil Method Number Color Result UnitAOCS Cc 13j-97 red 2 Unit AOCS Cc 13j-97 yellow 27 Unit

RBD469 oil was subjected to transesterification to produce fatty acidmethyl esters (FAMEs). The resulting FAME profile of RBD469 is shown inTable 20.

TABLE 20 FAME Profile of RBD469 Oil Fatty Acid Area % C10 0.01 C12:00.04 C14:0 0.64 C15:0 0.08 C16:0 8.17 C16:1 iso 0.39 C16:1 0.77 C17:00.08 C18:0 1.93 C18:1 85.88 C18:1 iso 0.05 C18:2 0.05 C20:0 0.3 C20:10.06 C20:1 0.44 C22:0 0.11 C23:0 0.03 C24:0 0.1 Total FAMEs Identified99.13

The oil stability indexes (OSI) of 6 RBD oil samples withoutsupplemented antioxidants and 3 RBD oil samples supplemented withantioxidants were analyzed according to the Oil Stability Index AOCSMethod Cd 12b-92. Shown in Table 21 are the results of OSI AOCS Cd12b-92 tests, conducted at 110° C., performed using a Metrohm 873Biodiesel Rancimat. Results, except where indicated with an astericks(*), are the average of multiple OSI runs. Those samples not analyzedare indicated (NA).

TABLE 21 Oil Stability Index at 110° C. of RBD oil samples with andwithout antioxidants Antioxidant Antioxidant OSI (hours) for each RBDSample added Concentration RBD437 RBD469 RBD502 RBD501 RBD503 RBD529None 0 65.41 38.33 72.10 50.32 63.04 26.68 Tocopherol 35 ppm/16.7 77.7248.60 82.67 NA NA NA & Ascorbyl ppm Palmitate Tocopherol 140 ppm/66.7130.27   81.54* 211.49* NA NA NA & Ascorbyl ppm Palmitate Tocopherol1050 ppm/500 >157*     >144      242.5* NA NA NA & Ascorbyl ppmPalmitate Tocopherol 50 ppm NA 46.97 NA NA NA NA TBHQ 20 ppm 63.37 37.4 NA NA NA NA

The untransformed P. moriformis (UTEX 1435) acid profile comprises lessthan 60% C18:1 fatty acids and greater than 7% C18:2 fatty acids. Incontrast, Strain D (comprising pSZ1500) exhibited fatty acid profileswith an increased composition of C18:1 fatty acids (to above 85%) and adecrease in C18:2 fatty acids (to less than 0.06%). Upon refining,bleaching, and degumming, RBD oils samples prepared from the oil madefrom strain E exhibited OSI values >26 hrs. With addition ofantioxidants, the OSI of RBD oils prepared from oils of Strain Dincreased from 48.60 hours to greater than 242 hours.

Example 8 Improving the Levels of Oleic Acid of Engineered MicrobesThrough Allelic Disruption of a Fatty Acid Desaturase and an Acyl-ACPThioesterase

This example describes the use of a transformation vector to disrupt aFATA locus of a Prototheca moriformis strain previously engineered forhigh oleic acid and low linoleic acid production. The transformationcassette used in this example comprised a selectable marker andnucleotide sequences encoding a P. moriformis KASII enzyme to engineermicroorganisms in which the fatty acid profile of the transformedmicroorganism has been altered for further increased oleic acid andlowered palmitic acid levels.

Strain D, described in Example 7 and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 137) according to biolistic transformationmethods in Example 2 and as described in PCT/U52009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. This strain was used as the host for transformationwith construct pSZ2276 to increase expression of a KASII enzyme whileconcomitantly ablating an endogenous acyl-ACP thioesterase genetic locusto generate Strain E. The pSZ2276 transformation construct included 5′(SEQ ID NO: 140) and 3′ (SEQ ID NO: 141) homologous recombinationtargeting sequences (flanking the construct) to the FATA1 genomic regionfor integration into the P. moriformis nuclear genome, an A. thalianaTHIC protein coding region under the control of the C. protothecoidesactin promoter/5′UTR (SEQ ID NO: 142) and C. vulgaris nitrate reductase3′ UTR (SEQ ID NO: 126). This AtTHIC expression cassette is listed asSEQ ID NO: 143 and served as a selection marker. The P. moriformis KASIIprotein coding region was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 128) and C. vulgaris nitrate reductase 3′UTR,and the native transit peptide of the KASII enzyme was replaced with theC. protothecoides stearoyl-ACP desaturase transit peptide (SEQ ID NO:129). The codon-optimized sequence of PmKASII comprising a C.protothecoides S106 stearoyl-ACP desaturase transit peptide is providedthe sequence listings as SEQ ID NO: 144. SEQ ID NO: 145 provides theprotein translation of SEQ ID NO: 144. The protein coding regions ofPmKASII and suc2 were codon optimized to reflect the codon bias inherentin P. moriformis UTEX 1435 nuclear genes as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ2276 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and a single engineered line,strain E was selected for analysis. Strain E was cultivated underheterotrophic lipid production conditions at pH5.0 and pH7.0 asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 3. The fatty acid profiles (expressed as Area % oftotal fatty acids) from the transgenic line arising from transformationwith pSZ2276 into Strain D are shown in Table 22.

TABLE 22 Fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and E engineered for increased oleic acid and loweredlinoleic acid levels. Transformation Area % Fatty Acid StrainConstruct(s) pH C16:0 C18:0 C18:1 C18:2 C20:1 Strain A None pH 5 26.63.3 60.5 6.7 0.07 Strain A None pH 7 28.3 4.1 58 6.5 0.06 Strain DpSZ1500 pH 5 17 3.6 77.1 0.01 0.14 Strain D pSZ1500 pH 7 19.5 5.3 72.60.01 0.09 Strain E pSZ1500 + pH 5 4.1 2.36 88.5 0.04 3.1 pSZ2276 StrainE pSZ1500 + pH 7 2.1 7.8 87.9 0.01 0.5 pSZ2276

As shown in Table 22, targeted interruption of FADc alleles with a CtOTEexpression cassette impacted the fatty acid profiles of transformedmicroorganisms. Fatty acid profiles of Strain D (comprising the pSZ1500transformation vector) showed increased composition of C18:1 fatty acidswith a concomitant decrease in C16:0 and C18:2 fatty acids relative toStrain A. Subsequent transformation of Strain D with pSZ2276 tooverexpress a P. moriformis (UTEX 1435) KASII protein whileconcomitantly ablating a FATA genetic locus (thereby generating StrainE) resulted in still further impact on the fatty acid profiles of thetransformed microorganisms. Fatty acid profiles of Strain E showedincreased composition of C18:1 fatty acids, with a further decrease inC16:0 fatty acids relative to Strains A and D. Propagation of Strain Ein culture conditions at pH 7, to induce expression from the Amt03promoter, resulted in a fatty acid profile that was higher in C18:0 andC18:1 fatty acids and lower in C16:0 fatty acids, relative to the samestrain cultured at pH 5.

These data demonstrate the utility of multiple genetic modifications toimpact the fatty acid profile of a host organism for increased levels ofoleic acid with concomitant decreased levels of linoleic acid andpalmitic acid. Further, this example illustrates the use of recombinantpolynucleotides to target gene interruption of an endogenous FATA allelewith a cassette comprising a pH-regulatable promoter to controlexpression of an exogenous KASII protein-coding region in order to alterthe fatty acid profile of a host microbe.

Example 9 Conditional Expression of a Fatty Acid Desaturase

This example describes the use of a transformation vector toconditionally express a delta 12 fatty acid desaturase (FAD) in aPrototheca moriformis strain previously engineered for high oleic acidand very low linoleic acid production in both seed and lipidproductivity stages of propagation. Very low linoleic acid levels innatural oils are sought for use in certain applications. However,absence of linoleic acid during cell division phase (“seed stage”) of ahost microbe is disadvantageous. Linoleic acid may be supplemented tothe seed medium to hasten cell division and not added during lipidproduction, but this addition imposes unwanted costs. To overcome thischallenge, a transformation cassette was constructed for regulatedexpression of a FAD2 enzyme such that levels of linoleic acidssufficient for cell division could be achieved and oil with very lowlevels of linoleic acids could be produced during the oil productionphase of culture of a microorgansim. The transformation cassette used inthis example comprised a selectable marker, a pH-regulatable promoter,and nucleotide sequences encoding a P. moriformis FAD2 enzyme toengineer microorganisms in which the fatty acid profile of thetransformed microorganism has been altered for increased oleic acidproduction and regulatable linoleic acid production.

Strain D, described in Examples 7 and 8 and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 137) according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strainwas used as the host for transformation with construct pSZ2413 tointroduce a pH-driven promoter for regulation of a P. moriformis FAD2enzyme. The pSZ2413 transformation construct included 5′ (SEQ ID NO:121) and 3′ (SEQ ID NO: 122) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the P. moriformis nuclear genome, an A. thaliana THICprotein coding region under the control of the C. protothecoides actinpromoter/5′UTR (SEQ ID NO: 142) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 126). This AtTHIC expression cassette is listed as SEQ IDNO: 143 and served as a selection marker. The P. moriformis FAD2 proteincoding region was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 128) and C. vulgaris nitrate reductase 3′UTR.The codon-optimized sequence of PmFAD2 is provided the sequence listingsas SEQ ID NO: 146. SEQ ID NO: 147 provides the protein translation ofSEQ ID NO: 146. The protein coding regions of PmFAD2 and suc2 were codonoptimized to reflect the codon bias inherent in P. moriformis UTEX 1435nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ2413 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and isolates of the engineeredline, Strain F were selected for analysis. These isolates werecultivated under heterotrophic lipid production conditions at pH7.0 (toactivate expression of FAD2 from the PmAmt03 promoter) and at pH5.0, asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 3. The resulting profile of C18:2 fatty acids(expressed in Area %) from nine representative isolates of transgenicStrain F (F-1 through F-9) arising from transformation with pSZ2413 intoStrain D are shown in Table 23.

TABLE 23 C18:2 fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and F. Area % C18:2 Strain Transformation Construct (s) pH5.0 pH 7.0 A None 6.07 7.26 D pSZ1500 0.01 0.01 F-1 pSZ1500 + pSZ24130.37 5.29 F-2 pSZ1500 + pSZ2413 0.45 6.87 F-3 pSZ1500 + pSZ2413 0.506.79 F-4 pSZ1500 + pSZ2413 0.57 5.06 F-5 pSZ1500 + pSZ2413 0.57 7.58 F-6pSZ1500 + pSZ2413 0.60 6.88 F-7 pSZ1500 + pSZ2413 0.62 6.52 F-8pSZ1500 + pSZ2413 0.63 5.79 F-9 pSZ1500 + pSZ2413 0.77 4.53

As shown in Table 23 the impact of regulated expression of the PmFAD2enzyme, effected though strain culture at different pH levels, is aclear increase in the composition of C18:2 fatty acids in thetransformed microorganism. Linoleic acid comprises about 6% to about7.3% of fatty acids of Strain A. In contrast, Strain D (comprising thepSZ1500 transformation vector to ablate both FAD2 alleles) ischaracterized by a fatty acid profile of 0.01% linoleic acid.Transformation of Strain D with pSZ2413 to generate Strain F results ina recombinant microbe in which the production of linoleic acid isregulated by the Amt03 promoter. Propagation of Strain F isolates inculture conditions at pH 7, to induce FAD2 expression from the Amt03promoter, resulted in a fatty acid profile characterized by about 4.5%to about 7.5% linoleic acid. In contrast, propagation of Strain Fisolates in culture conditions at pH 5 resulted in a fatty acid profilecharacterized by about 0.33 to about 0.77% linoleic acid.

These data demonstrate the utility of and effectiveness of recombinantpolynucleotides permitting conditional expression of a FAD2 enzyme toalter the fatty acid profile of engineered microorganisms, and inparticular in regulating the production of C18:2 fatty acids inmicrobial cells.

Examples 10-33 Introduction and Tables

Examples 10-33 below describe the engineering of various microorganismsin accordance with the present invention. To alter the fatty acidprofile of a microorganism, microorganisms may be genetically modifiedwherein endogenous or exogenous lipid biosynthesis pathway enzymes areexpressed, overexpressed, or attenuated. Steps to genetically engineer amicrobe to alter its fatty acid profile as to the degree of fatty acidunsaturation and to decrease or increase fatty acid chain lengthcomprise the design and construction of a transformation vector (e.g., aplasmid), transformation of the microbe with one or more vectors,selection of transformed microbes (transformants), growth of thetransformed microbe, and analysis of the fatty acid profile of thelipids produced by the engineered microbe.

Transgenes that alter the fatty acid profiles of host organisms may beexpressed in numerous eukaryotic microbes. Examples of expression oftransgenes in eukaryotic microbes including Chlamydomonas reinhardtii,Chlorella elhpsoidea, Chlorella protothecoides, Chlorella saccarophila,Chlorella vulgaris, Chlorella kessleri, Chlorella sorokiniana,Haematococcus pluvialis, Gonium pectorals, Volvox carteri, Dunaliellatertiolecta, Dunaliella viridis, Dunaliella salina, Closteriumperacerosum-strigosum-littorale complex, Nannochloropsis sp.,Thalassiosira pseudonana, Phaeodactylum tricornutum, Naviculasaprophila, Cylindrotheca fusiformis, Cyclotella cryptica, Symbiodiniummicroadriacticum, Amphidinium sp., Chaetoceros sp., Mortierella alpina,and Yarrowia hpolytica may be found in the scientific literature. Theseexpression techniques may be combined with the teachings of the presentinvention to produce engineered microorganisms with altered fatty acidprofiles.

Transgenes that alter the fatty acid profiles of host organisms or alterthe regiospecific distribution of glycerolipds produced by hostorganisms can also be expressed in numerous prokaryotic microbes.Examples of expression of transgenes in oleaginous microbes includingRhodococcus opacus may be found in the literature. These expressiontechniques may be combined with the teachings of the present inventionto produce engineered microorganisms with altered fatty acid profiles.

Tables 24A-E. Codon Preference Listing

TABLE 24A Amino Chlorella Chlorella Chlorella Chlorella DunaliellaVolvox Haematococcus Acid Codon sorokiniana vulgaris ellipsoideakessleri tertiolecta carteri pluvialis Ala GCG 0.20 0.25 0.15 0.14 0.090.25 0.21 Ala GCA 0.05 0.24 0.32 0.10 0.17 0.13 0.27 Ala GCT 0.12 0.160.26 0.18 0.31 0.26 0.17 Ala GCC 0.63 0.35 0.27 0.58 0.43 0.36 0.35 ArgAGG 0.03 0.09 0.10 0.09 0.26 0.08 0.14 Arg AGA 0.04 0.05 0.14 0.01 0.090.03 0.05 Arg CGG 0.06 0.19 0.09 0.06 0.06 0.17 0.15 Arg CGA 0.00 0.100.08 0.00 0.08 0.08 0.10 Arg CGT 0.06 0.09 0.37 0.14 0.12 0.22 0.13 ArgCGC 0.81 0.48 0.22 0.71 0.40 0.43 0.42 Asn AAT 0.04 0.16 0.43 0.06 0.270.23 0.21 Asn AAC 0.96 0.84 0.57 0.94 0.73 0.77 0.79 Asp GAT 0.13 0.250.47 0.12 0.40 0.35 0.27 Asp GAC 0.87 0.75 0.53 0.88 0.60 0.65 0.73 CysTGT 0.06 0.13 0.43 0.09 0.20 0.17 0.27 Cys TGC 0.94 0.87 0.57 0.91 0.800.83 0.64 End TGA 0.00 0.72 0.14 0.14 0.36 0.24 0.70 End TAG 0.33 0.110.29 0.00 0.00 0.18 0.22 End TAA 0.67 0.17 4.00 0.86 0.64 0.59 0.09 GlnCAG 0.42 0.40 0.15 0.40 0.27 0.29 0.33 Gln CAA 0.04 0.04 0.21 0.40 0.270.07 0.10 Glu GAG 0.53 0.50 0.33 0.40 0.27 0.53 0.49 Glu GAA 0.02 0.060.31 0.40 0.27 0.11 0.07 Gly GGG 0.04 0.16 0.19 0.08 0.10 0.12 0.22 GlyGGA 0.02 0.11 0.13 0.07 0.13 0.12 0.11 Gly GGT 0.03 0.12 0.39 0.24 0.250.23 0.15 Gly GGC 0.91 0.61 0.29 0.96 0.51 0.53 0.52 His CAT 0.14 0.160.30 0.08 0.25 0.35 0.27 His CAC 0.86 0.84 0.70 0.93 0.75 0.65 0.73 IleATA 0.00 0.04 0.07 0.01 0.04 0.08 0.09 Ile ATT 0.15 0.30 0.63 0.29 0.310.35 0.29 Ile ATC 0.85 0.66 0.65 0.69 0.65 0.57 0.62 Leu TTG 0.03 0.070.03 0.05 0.14 0.14 0.16 Leu TTA 0.00 0.01 0.32 0.00 0.02 0.03 0.02 LeuCTG 0.72 0.61 0.34 0.61 0.60 0.45 0.53 Leu CTA 0.01 0.03 0.03 0.04 0.040.07 0.07 Leu CTT 0.04 0.08 0.16 0.06 0.06 0.14 0.09 Leu CTC 0.20 0.200.12 0.24 0.14 0.17 0.13 Lys AAG 0.98 0.94 0.54 0.98 0.90 0.90 0.84 LysAAA 0.02 0.06 0.46 0.02 0.10 0.10 0.16 Met ATG 1.00 1.00 1.00 1.00 1.001.00 1.00 Phe TTT 0.28 0.32 0.42 0.31 0.24 0.27 0.35 Phe TTC 0.72 0.680.58 0.69 0.76 0.73 0.65 Pro CCG 0.18 0.31 0.09 0.07 0.04 0.34 0.15 ProCCA 0.06 0.17 0.36 0.07 0.04 0.20 0.24 Pro CCT 0.10 0.14 0.25 0.17 0.040.19 0.29 Pro CCC 0.66 0.38 0.29 0.69 0.04 0.27 0.32 Ser AGT 0.03 0.040.14 0.02 0.08 0.08 0.07 Ser AGC 0.27 0.38 0.18 0.18 0.31 0.27 0.31 SerTCG 0.12 0.14 0.08 0.10 0.02 0.19 0.10 Ser TCA 0.03 0.08 0.14 0.08 0.090.09 0.14 Ser TCT 0.09 0.11 0.26 0.18 0.19 0.14 0.13 Ser TCC 0.47 0.240.20 0.44 0.30 0.24 0.24 Thr ACG 0.11 0.20 0.13 0.05 0.12 0.27 0.19 ThrACA 0.01 0.20 0.32 0.07 0.20 0.12 0.23 Thr ACT 0.12 0.13 0.29 0.12 0.240.20 0.18 Thr ACC 0.76 0.47 0.26 0.76 0.44 0.41 0.40 Trp TGG 1.00 1.001.00 1.00 1.00 1.00 1.00 Tyr TAT 0.07 0.15 0.43 0.27 0.28 0.24 0.19 TyrTAC 0.93 0.85 0.57 0.73 0.72 0.76 0.81 Val GTG 0.71 0.54 0.37 0.60 0.540.46 0.62 Val GTA 0.00 0.05 0.25 0.03 0.09 0.07 0.09 Val GTT 0.11 0.140.24 0.09 0.14 0.17 0.09 Val GTC 0.18 0.27 0.14 0.28 0.23 0.30 0.21

TABLE 24B Closterium peracerosum- strigosum- Amino littorale DunaliellaDunaliella Gonium Phaeodactylum Chaetoceros Acid Codon complex viridissalina pectorale tricornutum compressum Ala GCG 0.48 0.13 0.15 0.43 0.150.08 Ala GCA 0.10 0.27 0.20 0.09 0.10 0.37 Ala GCT 0.15 0.25 0.27 0.080.23 0.36 Ala GCC 0.26 0.35 0.39 0.41 0.52 0.18 Arg AGG 0.04 0.25 0.220.13 0.02 0.14 Arg AGA 0.00 0.06 0.05 0.00 0.04 0.29 Arg CGG 0.18 0.080.12 0.40 0.10 0.00 Arg CGA 0.00 0.06 0.06 0.05 0.12 0.19 Arg CGT 0.130.15 0.13 0.08 0.41 0.38 Arg CGC 0.64 0.39 0.43 0.35 0.31 0.00 Asn AAT0.04 0.17 0.23 0.07 0.30 0.58 Asn AAC 0.96 0.83 0.77 0.93 0.65 0.42 AspGAT 0.30 0.38 0.40 0.11 0.41 0.53 Asp GAC 0.70 0.62 0.60 0.89 0.59 0.47Cys TGT 0.06 0.24 0.17 0.20 0.39 0.44 Cys TGC 0.94 0.76 0.83 0.90 0.610.56 End TGA 0.75 0.31 0.37 0.50 0.06 0.50 End TAG 0.00 0.15 0.14 0.000.13 0.00 End TAA 0.25 0.54 0.49 0.50 0.81 0.50 Gln CAG 0.53 0.36 0.320.31 0.23 0.16 Gln CAA 0.09 0.12 0.08 0.07 0.14 0.19 Glu GAG 0.31 0.440.51 0.56 0.21 0.28 Glu GAA 0.06 0.09 0.09 0.07 0.42 0.37 Gly GGG 0.310.14 0.10 0.18 0.08 0.12 Gly GGA 0.06 0.11 0.12 0.09 0.34 0.33 Gly GGT0.09 0.22 0.22 0.07 0.30 0.39 Gly GGC 0.53 0.54 0.56 0.65 0.28 0.16 HisCAT 0.33 0.25 0.25 0.43 0.28 0.84 His CAC 0.67 0.75 0.75 0.57 0.72 0.16Ile ATA 0.03 0.03 0.03 0.07 0.03 0.12 Ile ATT 0.23 0.25 0.31 0.33 0.510.65 Ile ATC 0.74 0.72 0.66 0.59 0.46 0.23 Leu TTG 0.04 0.11 0.12 0.040.26 0.11 Leu TTA 0.00 0.01 0.01 0.00 0.02 0.14 Leu CTG 0.31 0.60 0.610.64 0.15 0.05 Leu CTA 0.01 0.05 0.04 0.01 0.05 0.08 Leu CTT 0.04 0.070.08 0.05 0.18 0.51 Leu CTC 0.60 0.16 0.14 0.26 0.34 0.11 Lys AAG 0.860.87 0.89 0.93 0.75 0.52 Lys AAA 0.14 0.13 0.11 0.07 0.25 0.48 Met ATG1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.09 0.25 0.29 0.10 0.44 0.65 PheTTC 0.91 0.75 0.71 0.90 0.56 0.35 Pro CCG 0.28 0.10 0.08 0.53 0.29 0.05Pro CCA 0.15 0.10 0.17 0.09 0.12 0.45 Pro CCT 0.12 0.10 0.30 0.04 0.200.33 Pro CCC 0.44 0.10 0.45 0.34 0.40 0.17 Ser AGT 0.04 0.09 0.06 0.020.12 0.14 Ser AGC 0.05 0.31 0.32 0.20 0.12 0.07 Ser TCG 0.22 0.04 0.060.42 0.19 0.08 Ser TCA 0.16 0.08 0.10 0.09 0.06 0.31 Ser TCT 0.05 0.170.15 0.07 0.15 0.23 Ser TCC 0.47 0.31 0.30 0.20 0.35 0.18 Thr ACG 0.300.16 0.13 0.42 0.23 0.10 Thr ACA 0.06 0.21 0.18 0.03 0.13 0.38 Thr ACT0.22 0.18 0.23 0.08 0.19 0.27 Thr ACC 0.42 0.46 0.46 0.47 0.45 0.25 TrpTGG 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.07 0.16 0.21 0.12 0.18 0.67Tyr TAC 0.93 0.84 0.79 0.88 0.82 0.33 Val GTG 0.50 0.64 0.62 0.57 0.220.30 Val GTA 0.02 0.03 0.05 0.04 0.09 0.27 Val GTT 0.06 0.11 0.11 0.040.22 0.10 Val GTC 0.42 0.22 0.23 0.35 0.47 0.33

TABLE 24C Symbiodi- Nanno- Amino Cylindrotheca Amphidinium niummicro-chloropsis Cyclotella Navicula Thalassiosira C. Acid Codon fusiformiscarterae adriacticum sp cryptica pelliculosa pseudonana reinhardtii AlaGCG 0.07 0.17 0.22 0.24 0.11 0.00 0.11 0.35 Ala GCA 0.14 0.33 0.26 0.100.16 0.13 0.25 0.08 Ala GCT 0.35 0.29 0.20 0.17 0.45 0.44 0.33 0.13 AlaGCC 0.43 0.20 0.32 0.48 0.27 0.44 0.30 0.43 Arg AGG 0.09 0.15 0.27 0.000.09 0.05 0.18 0.05 Arg AGA 0.14 0.03 0.27 0.00 0.05 0.10 0.17 0.01 ArgCGG 0.06 0.08 0.09 0.00 0.04 0.05 0.06 0.20 Arg CGA 0.16 0.18 0.09 0.290.08 0.35 0.11 0.04 Arg CGT 0.34 0.18 0.09 0.14 0.47 0.20 0.34 0.09 ArgCGC 0.22 0.40 0.18 0.57 0.28 0.25 0.15 0.62 Asn AAT 0.42 0.37 0.21 0.000.25 0.47 0.43 0.09 Asn AAC 0.58 0.63 0.79 1.00 0.75 0.53 0.57 0.91 AspGAT 0.54 0.54 0.50 0.20 0.52 0.20 0.56 0.14 Asp GAC 0.46 0.46 0.50 0.800.48 0.80 0.44 0.86 Cys TGT 0.44 0.75 0.50 0.00 0.29 0.10 0.54 0.10 CysTGC 0.56 0.25 0.50 1.00 0.71 0.90 0.46 0.90 End TGA 0.13 0.50 1.00 0.000.10 0.00 0.31 0.27 End TAG 0.10 0.00 0.00 0.00 0.00 0.00 0.38 0.22 EndTAA 0.77 0.50 0.00 1.00 0.90 1.00 0.31 0.52 Gln CAG 0.12 0.33 0.28 0.410.19 0.21 0.16 0.38 Gln CAA 0.25 0.15 0.17 0.00 0.17 0.28 0.19 0.04 GluGAG 0.23 0.41 0.50 0.59 0.38 0.17 0.40 0.55 Glu GAA 0.39 0.10 0.06 0.000.26 0.34 0.26 0.03 Gly GGG 0.06 0.19 0.32 0.10 0.10 0.03 0.12 0.11 GlyGGA 0.47 0.10 0.12 0.05 0.45 0.28 0.51 0.06 Gly GGT 0.35 0.34 0.16 0.250.22 0.13 0.23 0.11 Gly GGC 0.12 0.37 0.40 0.60 0.24 0.56 0.14 0.72 HisCAT 0.39 0.12 0.40 0.00 0.42 1.00 0.50 0.11 His CAC 0.61 0.88 0.60 1.000.58 0.00 0.50 0.89 Ile ATA 0.06 0.05 0.00 0.00 0.04 0.00 0.08 0.03 IleATT 0.42 0.53 0.38 0.14 0.53 0.73 0.38 0.22 Ile ATC 0.52 0.42 0.63 0.860.42 0.27 0.54 0.75 Leu TTG 0.26 0.35 0.39 0.22 0.20 0.16 0.29 0.04 LeuTTA 0.09 0.01 0.00 0.00 0.03 0.00 0.05 0.01 Leu CTG 0.09 0.22 0.39 0.090.06 0.12 0.08 0.73 Leu CTA 0.05 0.00 0.04 0.00 0.03 0.04 0.06 0.03 LeuCTT 0.37 0.31 0.13 0.04 0.39 0.36 0.20 0.05 Leu CTC 0.13 0.12 0.04 0.650.29 0.32 0.32 0.15 Lys AAG 0.60 0.93 0.85 1.00 0.70 0.83 0.76 0.95 LysAAA 0.40 0.07 0.15 0.00 0.30 0.17 0.24 0.05 Met ATG 1.00 1.00 1.00 1.001.00 1.00 1.00 1.00 Phe TTT 0.37 0.21 0.25 0.20 0.31 0.78 0.38 0.16 PheTTC 0.63 0.79 0.75 0.80 0.69 0.22 0.62 0.84 Pro CCG 0.11 0.14 0.18 0.080.10 0.21 0.16 0.33 Pro CCA 0.33 0.42 0.09 0.08 0.16 0.29 0.31 0.08 ProCCT 0.32 0.22 0.41 0.25 0.35 0.21 0.31 0.13 Pro CCC 0.24 0.22 0.32 0.580.39 0.29 0.23 0.47 Ser AGT 0.12 0.13 0.09 0.00 0.09 0.13 0.18 0.04 SerAGC 0.09 0.24 0.14 0.13 0.08 0.28 0.11 0.35 Ser TCG 0.13 0.03 0.05 0.000.15 0.25 0.17 0.25 Ser TCA 0.12 0.25 0.05 0.00 0.12 0.08 0.12 0.05 SerTCT 0.30 0.16 0.23 0.13 0.39 0.25 0.23 0.07 Ser TCC 0.24 0.19 0.45 0.750.18 0.03 0.19 0.25 Thr ACG 0.09 0.14 0.10 0.28 0.10 0.18 0.21 0.30 ThrACA 0.15 0.28 0.10 0.00 0.15 0.09 0.19 0.08 Thr ACT 0.39 0.12 0.10 0.170.33 0.41 0.28 0.10 Thr ACC 0.37 0.47 0.70 0.56 0.43 0.32 0.32 0.52 TrpTGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.38 0.32 0.20 0.000.38 0.20 0.39 0.10 Tyr TAC 0.62 0.68 0.80 1.00 0.62 0.80 0.61 0.90 ValGTG 0.11 0.65 0.67 0.31 0.16 0.18 0.29 0.67 Val GTA 0.06 0.05 0.00 0.000.09 0.09 0.16 0.03 Val GTT 0.38 0.08 0.11 0.15 0.42 0.09 0.28 0.07 ValGTC 0.46 0.21 0.22 0.54 0.33 0.64 0.27 0.22

TABLE 24D Yarrowia Mortierella Rhodococcus Amino Acid Codon lipolyticaalpina opacus Ala GCG 0.08 0.14 0.35 Ala GCA 0.11 0.12 0.14 Ala GCT 0.350.29 0.09 Ala GCC 0.46 0.45 0.43 Arg AGG 0.05 0.05 0.05 Arg AGA 0.130.06 0.02 Arg CGG 0.12 0.06 0.26 Arg CGA 0.52 0.09 0.12 Arg CGT 0.110.32 0.11 Arg CGC 0.07 0.42 0.44 Asn AAT 0.17 0.15 0.21 Asn AAC 0.830.85 0.79 Asp GAT 0.35 0.42 0.24 Asp GAC 0.65 0.58 0.76 Cys TGT 0.460.13 0.26 Cys TGC 0.54 0.87 0.74 End TGA 0.16 0.05 0.72 End TAG 0.380.25 0.17 End TAA 0.46 0.70 0.11 Gln CAG 0.33 0.36 0.28 Gln CAA 0.080.06 0.06 Glu GAG 0.44 0.49 0.45 Glu GAA 0.14 0.09 0.22 Gly GGG 0.050.03 0.18 Gly GGA 0.28 0.29 0.15 Gly GGT 0.32 0.32 0.20 Gly GGC 0.340.36 0.48 His CAT 0.34 0.27 0.20 His CAC 0.66 0.73 0.80 Ile ATA 0.030.01 0.05 Ile ATT 0.44 0.33 0.14 Ile ATC 0.53 0.66 0.81 Leu TTG 0.090.27 0.09 Leu TTA 0.02 0.00 0.01 Leu CTG 0.37 0.26 0.41 Leu CTA 0.050.02 0.03 Leu CTT 0.18 0.12 0.06 Leu CTC 0.29 0.32 0.40 Lys AAG 0.840.91 0.80 Lys AAA 0.16 0.09 0.20 Met ATG 1.00 1.00 1.00 Phe TTT 0.380.39 0.09 Phe TTC 0.62 0.61 0.91 Pro CCG 0.10 0.07 0.52 Pro CCA 0.100.08 0.09 Pro CCT 0.32 0.36 0.07 Pro CCC 0.47 0.49 0.32 Ser AGT 0.070.05 0.08 Ser AGC 0.11 0.14 0.23 Ser TCG 0.16 0.32 0.33 Ser TCA 0.080.08 0.07 Ser TCT 0.28 0.12 0.05 Ser TCC 0.30 0.29 0.24 Thr ACG 0.110.17 0.28 Thr ACA 0.14 0.10 0.11 Thr ACT 0.26 0.23 0.07 Thr ACC 0.490.49 0.53 Trp TGG 1.00 1.00 1.00 Tyr TAT 0.18 0.20 0.18 Tyr TAC 0.820.80 0.82 Val GTG 0.33 0.22 0.37 Val GTA 0.05 0.02 0.05 Val GTT 0.260.27 0.10 Val GTC 0.36 0.49 0.49

TABLE 24E Preferred codon usage in Chlorella protothecoides TTC (Phe)TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg)CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met)AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)

TABLE 25 Lipid biosynthesis pathway proteins. 3-Ketoacyl ACP synthaseCuphea hookeriana 3-ketoacyl-ACP synthase (GenBank Acc. No. AAC68861.1),Cuphea wrightii beta-ketoacyl-ACP synthase II (GenBank Acc. No.AAB37271.1), Cuphea lanceolata beta-ketoacyl-ACP synthase IV (GenBankAcc. No. CAC59946.1), Cuphea wrightii beta-ketoacyl-ACP synthase II(GenBank Acc. No. AAB37270.1), Ricinus communis ketoacyl-ACP synthase(GenBank Acc. No. XP_002516228), Gossypium hirsutum ketoacyl- ACPsynthase (GenBank Acc. No. ADK23940.1), Glycine max plastid3-keto-acyl-ACP synthase II-A (GenBank Acc No. AAW88763.1), Elaeisguineensis beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAF26738.2),Helianthus annuus plastid 3-keto-acyl-ACP synthase I (GenkBank Acc. No.ABM53471.1), Glycine max3-keto-acyl-ACP synthase I (GenkBank Acc. No.NP_001238610.1), Helianthus annuus plastid 3-keto-acyl-ACP synthase II(GenBank Acc ABI18155.1), Brassica napus beta-ketoacyl-ACP synthetase 2(GenBank Acc. No. AAF61739.1), Perilla frutescens beta-ketoacyl-ACPsynthase II (GenBank Acc. No. AAC04692.1), Helianthus annusbeta-ketoacyl-ACP synthase II (GenBank Accession No. ABI18155), Ricinuscommunis beta-ketoacyl-ACP synthase II (GenBank Accession No. AAA33872),Haematococcus pluvialis beta-ketoacyl acyl carrier protein synthase(GenBank Accession No. HM560033.1), Jatropha curcasbeta ketoacyl-ACPsynthase I (GenBank Accession No. ABJ90468.1), Populus trichocarpabeta-ketoacyl-ACP synthase I (GenBank Accession No. XP_002303661.1),Coriandrum sativum beta-ketoacyl- ACP synthetase I (GenBank AccessionNo. AAK58535.1), Arabidopsis thaliana 3-oxoacyl- [acyl-carrier-protein]synthase I (GenBank Accession No. NP_001190479.1), Vitis vinifera 3-oxoacyl-[acyl-carrier-protein] synthase I (GenBank Accession No.XP_002272874.2) Fatty acyl-ACP Thioesterases Umbellularia californicafatty acyl-ACP thioesterase (GenBank Acc. No. AAC49001), Cinnamomumcamphora fatty acyl-ACP thioesterase (GenBank Acc. No. Q39473),Umbellularia californica fatty acyl-ACP thioesterase (GenBank Acc. No.Q41635), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc.No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBankAcc. No. AAB71730), Elaeis guineensis fatty acyl- ACP thioesterase(GenBank Acc. No. ABD83939), Elaeis guineensis fatty acyl-ACPthioesterase (GenBank Acc. No. AAD42220), Populus tomentosa fattyacyl-ACP thioesterase (GenBank Acc. No. ABC47311), Arabidopsis thalianafatty acyl-ACP thioesterase (GenBank Acc. No. NP_172327), Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank Acc. No. CAA85387),Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank Acc. No.CAA85388), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc.No. Q9SQI3), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank Acc.No. CAA54060), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBankAcc. No. AAC72882), Cuphea calophylla subsp. mesostemon fatty acyl-ACPthioesterase (GenBank Acc. No. ABB71581), Cuphea lanceolata fattyacyl-ACP thioesterase (GenBank Acc. No. CAC19933), Elaeis guineensisfatty acyl-ACP thioesterase (GenBank Acc. No. AAL15645), Cupheahookeriana fatty acyl- ACP thioesterase (GenBank Acc. No. Q39513),Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc. No.AAD01982), Vitis vinifera fatty acyl-ACP thioesterase (GenBank Acc. No.CAN81819), Garcinia mangostana fatty acyl-ACP thioesterase (GenBank Acc.No. AAB51525), Brassica juncea fatty acyl-ACP thioesterase (GenBank Acc.No. ABI18986), Madhuca longifolia fatty acyl-ACP thioesterase (GenBankAcc. No. AAX51637), Brassica napus fatty acyl-ACP thioesterase (GenBankAcc. No. ABH11710), Brassica napus fatty acyl-ACP thioesterase (GenBankAcc. No. CAA52070.1), Oryza sativa (indica cultivar-group) fattyacyl-ACP thioesterase (GenBank Acc. No. EAY86877), Oryza sativa(japonica cultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No.NP_001068400), Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank Acc. No. EAY99617), Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank Acc. No. AAC49269), Ulmus Americana fattyacyl-ACP thioesterase (GenBank Acc. No. AAB71731), Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank Acc. No. CAB60830), Cupheapalustris fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49180), Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858, Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858.1),Cuphea palustris fatty acyl-ACP thioesterase (GenBank Acc. No.AAC49179), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc.No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBankAcc. No. AAB717291.1), Cuphea hookeriana fatty acyl-ACP thioesteraseGenBank Acc. No. U39834), Umbelluaria californica fatty acyl-ACPthioesterase (GenBank Acc. No. M94159), Cinnamomum camphora fattyacyl-ACP thioesterase (GenBank Acc. No. U31813), Ricinus communis fattyacyl-ACP thioesterase (GenBank Acc. No. ABS30422.1), Helianthus annuusacyl-ACP thioesterase (GenBank Accession No. AAL79361.1), Jatrophacurcas acyl-ACP thioesterase (GenBank Accession No. ABX82799.3), Zeamays oleoyl-acyl carrier protein thioesterase, (GenBank Accession No.ACG40089.1), Haematococcus pluvialis fatty acyl- ACP thioesterase(GenBank Accession No. HM560034.1) Desaturase Enzymes Linumusitatissimum fatty acid desaturase 3C, (GenBank Acc. No. ADV92272.1),Ricinus communis omega-3 fatty acid desaturase, endoplasmic reticulum,putative, (GenBank Acc. No. EEF36775.1), Vernicia fordii omega-3 fattyacid desaturase, (GenBank Acc. No. AAF12821), Glycine max chloroplastomega 3 fatty acid desaturase isoform 2, (GenBank Acc. No. ACF19424.1),Prototheca moriformis FAD-D omega 3 desaturase (SEQ ID NO: 35),Prototheca moriformis linoleate desaturase (SEQ ID NO: 36), Carthamustinctorius delta 12 desaturase, (GenBank Accession No. ADM48790.1),Gossypium hirsutum omega-6 desaturase, (GenBank Accession No.CAA71199.1), Glycine max microsomal desaturase (GenBank Accession No.BAD89862.1), Zea mays fatty acid desaturase (GenBank Accession No.ABF50053.1), Brassica napa linoleic acid desaturase (GenBank AccessionNo. AAA32994.1), Camelina sativa omega-3 desaturase (SEQ ID NO: 37),Prototheca moriformis delta 12 desaturase allele 2 (SEQ ID NO: 38,Camelina sativa omega-3 FAD7-1 (SEQ ID NO: 159), Helianthus annuusstearoyl-ACP desaturase, (GenBank Accession No. AAB65145.1), Ricinuscommunis stearoyl-ACP desaturase, (GenBank Accession No. AACG59946.1),Brassica juncea plastidic delta-9-stearoyl-ACP desaturase (GenBankAccession No. AAD40245.1), Glycine max stearoyl-ACP desaturase (GenBankAccession No. ACJ39209.1), Olea europaea stearoyl-ACP desaturase(GenBank Accession No. AAB67840.1), Vernicia fordiistearoyl-acyl-carrier protein desaturase, (GenBank Accession No.ADC32803.1), Descurainia sophia delta-12 fatty acid desaturase (GenBankAccession No. ABS86964.2), Euphorbia lagascae delta12-oleic aciddesaturase (GenBank Acc. No. AAS57577.1), Chlorella vulgaris delta 12fatty acid desaturease (GenBank Accession No. ACF98528), Chlorellavulgaris omega-3 fatty acid desaturease (GenBank Accession No.BAB78717), Haematococcus pluvialis omega-3 fatty acid desaturase(GenBank Accession No. HM560035.1), Haematococcus pluvialisstearoyl-ACP-desaturase GenBank Accession No. EF586860.1, Haematococcuspluvialis stearoyl-ACP-desaturase GenBank Accession No. EF523479.1Oleate 12-hydroxylase Enzymes Ricinus communisoleate 12-hydroxylase(GenBank Acc. No. AAC49010.1), Physaria lindheimeri oleate12-hydroxylase (GenBank Acc. No. ABQ01458.1), Physaria lindheimerimutant bifunctional oleate 12-hydroxylase: desaturase (GenBank Acc. No.ACF17571.1), Physaria lindheimeri bifunctional oleate 12-hydroxylase:desaturase (GenBank Accession No. ACQ42234.1), Physaria lindheimeribifunctional oleate 12- hydroxylase: desaturase (GenBank Acc. No.AAC32155.1), Arabidopsis lyrata subsp. Lyrata (GenBank Acc. No.XP_002884883.1) Glycerol-3-phosphate Enzymes Arabidopsis thalianaglycerol-3-phosphate acyltransferase BAA00575, Chlamydomonas reinhardtiiglycerol-3-phosphate acyltransferase (GenBank Acc. No. EDP02129),Chlamydomonas reinhardtii glycerol-3-phosphate acyltransferase (GenBankAcc. No. Q886Q7), Cucurbita moschata acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GenBank Acc. No. BAB39688), Elaeisguineensis glycerol-3-phosphate acyltransferase, ((GenBank Acc. No.AAF64066), Garcina mangostana glycerol-3-phosphate acyltransferase(GenBank Acc. No. ABS86942), Gossypium hirsutum glycerol-3-phosphateacyltransferase (GenBank Acc. No. ADK23938), Jatropha curcasglycerol-3-phosphate acyltransferase (GenBank Acc. No. ADV77219),Jatropha curcas plastid glycerol-3- phosphate acyltransferase (GenBankAcc. No. ACR61638), Ricinus communis plastidial glycerol-phosphateacyltransferase (GenBank Acc. No. EEF43526), Vica faba glycerol-3-phosphate acyltransferase (GenBank Accession No. AAD05164), Zea maysglycerol-3- phosphate acyltransferase (GenBank Acc. No. ACG45812)Lysophosphatidic acid acyltransferase Enzymes Arabidopsis thaliana1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.AEE85783), Brassica juncea 1-acyl-sn-glycerol-3-phosphateacyltransferase (GenBank Accession No. ABQ42862), Brassica juncea1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.ABM92334), Brassica napus 1-acyl-sn-glycerol-3-phosphate acyltransferase(GenBank Accession No. CAB09138), Chlamydomonas reinhardtiilysophosphatidic acid acyltransferase (GenBank Accession No. EDP02300),Cocos nucifera lysophosphatidic acid acyltransferase (GenBank Acc. No.AAC49119), Limnanthes alba lysophosphatidic acid acyltransferase(GenBank Accession No. EDP02300), Limnanthes douglasii1-acyl-sn-glycerol-3-phosphate acyltransferase (putative) (GenBankAccession No. CAA88620), Limnanthes douglasii acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (GenBank Accession No.ABD62751), Limnanthes douglasii 1-acylglycerol-3-phosphate O-acyltransferase (GenBank Accession No. CAA58239), Ricinus communis1-acyl-sn-glycerol- 3-phosphate acyltransferase (GenBank Accession No.EEF39377) Diacylglycerol acyltransferase Enzymes Arabidopsis thalianadiacylglycerol acyltransferase (GenBank Acc. No. CAB45373), Brassicajuncea diacylglycerol acyltransferase (GenBank Acc. No. AAY40784),Elaeis guineensis putative diacylglycerol acyltransferase (GenBank Acc.No. AEQ94187), Elaeis guineensis putative diacylglycerol acyltransferase(GenBank Acc. No. AEQ94186), Glycine max acyl CoA: diacylglycerolacyltransferase (GenBank Acc. No. AAT73629), Helianthus annusdiacylglycerol acyltransferase (GenBank Acc. No. ABX61081), Oleaeuropaea acyl- CoA: diacylglycerol acyltransferase 1 (GenBank Acc. No.AAS01606), Ricinus communis diacylglycerol acyltransferase (GenBank Acc.No. AAR11479) Phospholipid diacylglycerol acyltransferase EnzymesArabidopsis thaliana phospholipid: diacylglycerol acyltransferase(GenBank Acc. No. AED91921), Elaeis guineensis putative phospholipid:diacylglycerol acyltransferase (GenBank Acc. No. AEQ94116), Glycine maxphospholipid: diacylglycerol acyltransferase 1-like (GenBank Acc. No.XP_003541296), Jatropha curcas phospholipid: diacylglycerolacyltransferase (GenBank Acc. No. AEZ56255), Ricinus communisphospholipid: diacylglycerol acyltransferase (GenBank Acc. No.ADK92410), Ricinus communis phospholipid: diacylglycerol acyltransferase(GenBank Acc. No. AEW99982)

Example 10 Engineering Chlorella sorokiniana

Expression of recombinant genes in accordance with the present inventionin Chlorella sorokiniana may be accomplished by modifying the methodsand vectors taught by Dawson et al. as discussed herein. Briefly, Dawsonet al., Current Microbiology Vol. 35 (1997) pp. 356-362, reported thestable nuclear transformation of Chlorella sorokiniana with plasmid DNA.Using the transformation method of microprojectile bombardment, Dawsonintroduced the plasmid pSV72-NRg, encoding the full Chlorella vulgarisnitrate reductase gene (NR, GenBank Accession No. U39931), into mutantChlorella sorokiniana (NR-mutants). The NR-mutants are incapable ofgrowth without the use of nitrate as a source of nitrogen. Nitratereductase catalyzes the conversion of nitrate to nitrite. Prior totransformation, Chlorella sorokiniana NR-mutants were unable to growbeyond the microcolony stage on culture medium comprising nitrate (NO₃⁻) as the sole nitrogen source. The expression of the Chlorella vulgarisNR gene product in NR-mutant Chlorella sorokiniana was used as aselectable marker to rescue the nitrate metabolism deficiency. Upontransformation with the pSV72-NRg plasmid, NR-mutant Chlorellasorokiniana stably expressing the Chlorella vulgaris NR gene productwere obtained that were able to grow beyond the microcolony stage onagar plates comprising nitrate as the sole carbon source. Evaluation ofthe DNA of the stable transformants was performed by Southern analysisand evaluation of the RNA of the stable transformants was performed byRNase protection. Selection and maintenance of the transformed Chlorellasorokiniana (NR mutant) was performed on agar plates (pH 7.4) comprising0.2 g/L MgSO₄, 0.67 g/L KH₂PO₄, 3.5 g/L K₂HPO₄, 1.0 g/L Na₃C₆H_(S)O₇.H₂Oand 16.0 g/L agar, an appropriate nitrogen source (e.g., NO₃),micronutrients, and a carbon source. Dawson also reported thepropagation of Chlorella sorokiniana and Chlorella sorokiniana NRmutants in liquid culture medium. Dawson reported that the plasmidpSV72-NRg and the promoter and 3′ UTR/terminator of the Chlorellavulgaris nitrate reductase gene were suitable to enable heterologousgene expression in Chlorella sorokiniana NR-mutants. Dawson alsoreported that expression of the Chlorella vulgaris nitrate reductasegene product was suitable for use as a selectable marker in Chlorellasorokiniana NR-mutants.

In an embodiment of the present invention, vector pSV72-NRg, comprisingnucleotide sequence encoding the Chlorella vulgaris nitrate reductase(CvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 25, eachprotein-coding sequence codon-optimized for expression in Chlorellasorokiniana to reflect the codon bias inherent in nuclear genes ofChlorella sorokiniana in accordance with Tables 24A-D. For each lipidbiosynthesis pathway protein of Table 25, the codon-optimized genesequence can individually be operably linked to the CvNR promoterupstream of the protein-coding sequence and operably linked to the CvNR3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chlorella sorokiniana genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorellasorokiniana with the transformation vector is achieved throughwell-known transformation techniques including microprojectilebombardment or other known methods. Activity of the CvNR gene productmay be used as a selectable marker to rescue the nitrogen assimiliationdeficiency of Chlorella sorokiniana NR mutant strains and to select forChlorella sorokiniana NR-mutants stably expressing the transformationvector. Growth media suitable for Chlorella sorokiniana lipid productioninclude, but are not limited to 0.5 g/L KH₂PO₄, 0.5 g/L K₂HPO₄, 0.25 g/LMgSO₄-7H2O, with supplemental micronutrients and the appropriatenitrogen and carbon sources (Patterson, Lipids Vol.5:7 (1970), pp.597-600). Evaluation of fatty acid profiles of Chlorella sorokinianalipids may be assessed through standard lipid extraction and analyticalmethods described herein.

Example 11 Engineering Chlorella vulgaris

Expression of recombinant genes in accordance with the present inventionin Chlorella vulgaris may be accomplished by modifying the methods andvectors taught by Chow and Tung et al. as discussed herein. Briefly,Chow and Tung et al., Plant Cell Reports, Volume 18 (1999), pp. 778-780,reported the stable nuclear transformation of Chlorella vulgaris withplasmid DNA. Using the transformation method of electroporation, Chowand Tung introduced the plasmid pIG121-Hm (GenBank Accession No.AB489142) into Chlorella vulgaris. The nucleotide sequence of pIG121-Hmcomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter upstream of the GUSprotein-coding sequence and further operably linked to the 3′UTR/terminator of the nopaline synthase (nos) gene downstream of the GUSprotein-coding sequence. The sequence of plasmid pIG121-Hm furthercomprised a hygromycin B antibiotic resistance cassette. This hygromycinB antibiotic resistance cassette comprised a CaMV 35S promoter operablylinked to sequence encoding the hygromycin phosphotransferase (hpt,GenBank Accession No. BAH24259) gene product. Prior to transformation,Chlorella vulgaris was unable to be propagated in culture mediumcomprising 50 ug/ml hygromycin B. Upon transformation with the pIG121-Hmplasmid, transformants of Chlorella vulgaris were obtained that werepropagated in culture medium comprising 50 ug/ml hyrgromycin B. Theexpression of the hpt gene product in Chlorella vulgaris enabledpropagation of transformed Chlorella vulgaris in the presence of 50ug/mL hyrgromycin B, thereby establishing the utility of the ahygromycin B resistance cassette as a selectable marker for use inChlorella vulgaris. Detectable activity of the GUS reporter geneindicated that CaMV 35S promoter and nos 3′UTR are suitable for enablingheterologous gene expression in Chlorella vulgaris. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. Selection and maintenance of transformed Chlorella vulgariswas performed on agar plates comprising YA medium (agar and 4 g/L yeastextract). The propagation of Chlorella vulgaris in liquid culture mediumwas conducted as discussed by Chow and Tung. Propagation of Chlorellavulgaris in media other than YA medium has been described (for examples,see Chader et al., Revue des Energies Renouvelabes, Volume 14 (2011),pp. 21-26 and Illman et al., Enzyme and Microbial Technology, Vol. 27(2000), pp. 631-635). Chow and Tung reported that the plasmid pIG121-Hm,the CaMV 35S promoter, and the Agrobacterium tumefaciens nopalinesynthase gene 3′UTR/terminator are suitable to enable heterologous geneexpression in Chlorella vulgaris. In addition, Chow and Tung reportedthe hyromycin B resistance cassette was suitable for use as a selectablemarker in Chlorella vulgaris. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Chlorella vulgaris have been discussedin Chader et al., Revue des Energies Renouvelabes, Volume 14 (2011), pp.21-26.

In an embodiment of the present invention, pIG121-Hm, comprising thenucleotide sequence encoding the hygromycin B gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Chlorella vulgaris to reflect thecodon bias inherent in nuclear genes of Chlorella vulgaris in accordancewith Tables 24A-D. For each lipid biosynthesis pathway protein of Table25, the codon-optimized gene sequence can individually be operablylinked to the CaMV 35S promoter upstream of the protein-coding sequenceand operably linked to the Agrobacterium tumefaciens nopaline synthasegene 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Chlorella vulgaris genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorella vulgariswith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the hygromycin B resistance gene product may beused as a marker to select for Chlorella vulgaris transformed with thetransformation vector on, but not limited to, agar medium comprisinghygromycin. Growth media suitable for Chlorella vulgaris lipidproduction include, but are not limited to BG11 medium (0.04 g/L KH₂PO₄,0.075 g/L CaCl₂, 0.036 g/L citric acid, 0.006 g/L Ammonium FerricCitrate, 1 mg/L EDTA, and 0.02 g/L Na₂CO₃) supplemented with tracemetals, and optionally 1.5 g/L NaNO3. Additional media suitable forculturing Chlorella vulgaris for lipid production include, for example,Watanabe medium (comprising 1.5 g/L KNO₃, 1.25 g/L KH₂PO₄, 1.25 g l⁻¹MgSO₄.7H₂O, 20 mg l⁻¹ FeSO₄.7H₂O with micronutrients and low-nitogenmedium (comprising 203 mg/l (NH₄)₂HPO₄, 2.236 g/l KCl, 2.465 g/l MgSO₄,1.361 g/l KH₂PO₄ and 10 mg/l FeSO₄) as reported by Illman et al., Enzymeand Microbial Technology, Vol. 27 (2000), pp. 631-635. Evaluation offatty acid profiles of Chlorella vulgaris lipids may be assessed throughstandard lipid extraction and analytical methods described herein.

Example 12 Engineering Chlorella ellipsoidea

Expression of recombinant genes in accordance with the present inventionin Chlorella ellipsoidea may be accomplished by modifying the methodsand vectors taught by Chen et al. as discussed herein. Briefly, Chen etal., Current Genetics, Vol. 39:5 (2001), pp. 365-370, reported thestable transformation of Chlorella ellipsoidea with plasmid DNA. Usingthe transformation method of electroporation, Chen introduced theplasmid pBinUΩNP-1 into Chlorella ellipsoidea. The nucleotide sequenceof pBinUΩNP-1 comprised sequence encoding the neutrophil peptide-1(NP-1) rabbit gene product operably linked to a Zea mays Ubiquitin(ubil) gene promoter upstream of the NP-1 protein-coding region andoperably linked to the 3′ UTR/terminator of the nopaline synthase (nos)gene downstream of the NP-1 protein-coding region. The sequence ofplasmid pBinUΩNP-1 further comprised a G418 antibiotic resistancecassette. This G418 antibiotic resistance cassette comprised sequenceencoding the aminoglycoside 3′-phosphotransferase (aph 3′) gene product.The aph 3′ gene product confers resistance to the antibiotic G418. Priorto transformation, Chlorella ellipsoidea was unable to be propagated inculture medium comprising 30 ug/mL G418. Upon transformation with thepBinUΩNP-1 plasmid, transformants of Chlorella ellipsoidea were obtainedthat were propagated in selective culture medium comprising 30 ug/mLG418. The expression of the aph 3′ gene product in Chlorella ellipsoideaenabled propagation of transformed Chlorella ellipsoidea in the presenceof 30 ug/mL G418, thereby establishing the utility of the G418antibiotic resistance cassette as selectable marker for use in Chlorellaellipsoidea. Detectable activity of the NP-1 gene product indicated thatthe ubil promoter and nos 3′ UTR are suitable for enabling heterologousgene expression in Chlorella ellipsoidea. Evaluation of the genomic DNAof the stable transformants was performed by Southern analysis.Selection and maintenance of the transformed Chlorella ellipsoidea wasperformed on Knop medium (comprising 0.2 g/L K₂HPO₄, 0.2 g/L MgSO₄.7H₂O,0.12 g/L KCl, and 10 mg/L FeCl3, pH 6.0-8.0 supplemented with 0.1% yeastextract and 0.2% glucose) with 15 ug/mL G418 (for liquid cultures) orwith 30 ug/mL G418 (for solid cultures comprising 1.8% agar).Propagation of Chlorella ellipsoidea in media other than Knop medium hasbeen reported (see Cho et al., Fisheries Science, Vol. 73:5 (2007), pp.1050-1056, Jarvis and Brown, Current Genetics, Vol. 19 (1991), pp.317-321 and Kim et al., Marine Biotechnology, Vol. 4 (2002), pp.63-'73). Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inChlorella ellipsoidea have been reported (see Jarvis and Brown and Kimet al., Marine Biotechnology, Vol. 4 (2002), pp. 63-'73). Chen reportedthat the plasmid pBinUΩNP-1, the ubil promoter, and the Agrobacteriumtumefaciens nopaline synthase gene 3′UTR/terminator are suitable toenable exogenous gene expression in Chlorella ellipsoidea. In addition,Chen reported that the G418 resistance cassette encoded on pBinUΩNP-1was suitable for use as a selectable marker in Chlorella ellipsoidea.

In an embodiment of the present invention, vector pBinUΩNP-1, comprisingthe nucleotide sequence encoding the aph 3′ gene product, conferringresistance to G418, for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 25, eachprotein-coding sequence codon-optimized for expression in Chlorellaellipsoidea to reflect the codon bias inherent in nuclear genes ofChlorella ellipsoidea in accordance with Tables 24A-D. For each lipidbiosynthesis pathway protein of Table 25, the codon-optimized genesequence can individually be operably linked to the Zea mays ubilpromoter upstream of the protein-coding sequence and operably linked tothe Agrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator atthe 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella ellipsoidea genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella ellipsoidea with the transformationvector is achieved through well-known transformation techniquesincluding electroporation or other known methods. Activity of the aph 3′gene product may be used as a marker to select for Chlorella ellipsoideatransformed with the transformation vector on, but not limited to, Knopagar medium comprising G418. Growth media suitable for Chlorellaellipsoidea lipid production include, but are not limited to, Knopmedium and those culture medium reported by Jarvis and Brown and Kim etal. Evaluation of fatty acid profiles of Chlorella ellipsoidea lipidsmay be assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 13 Engineering Chlorella kessleri

Expression of recombinant genes in accordance with the present inventionin Chlorella kessleri may be accomplished by modifying the methods andvectors taught by El-Sheekh et al. as discussed herein. Briefly,El-Sheekh et al., Biologia Plantarium, Vol. 42:2 (1999), pp. 209-216,reported the stable transformation of Chlorella kessleri with plasmidDNA. Using the transformation method of microprojectile bombardment,El-Sheekh introduced the plasmid pBI121 (GenBank Accession No. AF485783)into Chlorella kessleri. Plasmid pBI121 comprised a kanamycin/neomycinantibiotic resistance cassette. This kanamycin/neomycin antibioticresistance cassette comprised the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter, sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) for resistance to kanamycin and G418, and the 3′UTR/terminator of the Agrobacterium tumefaciens nopaline synthase (nos)gene. pBI121 further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably linked to a CaMV 35S promoter andoperably linked to a 3′ UTR/terminator of the nos gene. Prior totransformation, Chlorella kessleri was unable to be propagated inculture medium comprising 15 ug/L kanamycin. Upon transformation withthe pBI121plasmid, transformants of Chlorella kessleri were obtainedthat were propagated in selective culture medium comprising 15 mg/Lkanamycin. The express ion of the nptII gene product in Chlorellakessleri enabled propagation in the presence of 15 mg/L kanamycin,thereby establishing the utility of the kanamycin/neomycin antibioticresistance cassette as selectable marker for use in Chlorella kessleri.Detectable activity of the GUS gene product indicated that the CaMV 35Spromoter and nos 3′ UTR are suitable for enabling heterologous geneexpression in Chlorella kessleri. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. As reported byEl-Sheekh, selection and maintenance of transformed Chlorella kessleriwas conducted on semisolid agar plates comprising YEG medium (1% yeastextract, 1% glucose) and 15 mg/L kanamycin. El-Sheekh also reported thepropagation of Chlorella kessleri in YEG liquid culture media.Additional media suitable for culturing Chlorella kessleri for lipidproduction are disclosed in Sato et al., BBA Molecular and Cell Biologyof Lipids, Vol. 1633 (2003), pp. 27-34). El-Sheekh reported that theplasmid pBI121, the CaMV promoter, and the nopaline synthase gene3′UTR/terminator are suitable to enable heterologous gene expression inChlorella kessleri. In addition, El-Sheekh reported that thekanamycin/neomycin resistance cassette encoded on pBI121 was suitablefor use as a selectable marker in Chlorella kessleri.

In an embodiment of the present invention, vector pBI121, comprising thenucleotide sequence encoding the kanamycin/neomycin resistance geneproduct for use as a selectable marker, is constructed and modified tofurther comprise a lipid biosynthesis pathway expression cassettesequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 25, eachprotein-coding sequence codon-optimized for expression in Chlorellakessleri to reflect the codon bias inherent in nuclear genes ofChlorella kessleri in accordance with Tables 24A-D. For each lipidbiosynthesis pathway protein of Table 25, the codon-optimized genesequence can individually be operably linked to the CaMV 35S promoterupstream of the protein-coding sequence and operably linked to theAgrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella kessleri genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella kessleri with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nptII gene product may be used as a marker to select forChlorella kessleri transformed with the transformation vector on, butnot limited to, YEG agar medium comprising kanamycin or neomycin. Growthmedia suitable for Chlorella kessleri lipid production include, but arenot limited to, YEG medium, and those culture media reported by Sato etal. Evaluation of fatty acid profiles of Chlorella kessleri lipids maybe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 14 Engineering Dunaliella tertiolecta

Expression of recombinant genes in accordance with the present inventionin Dunaliella tertiolecta may be accomplished by modifying the methodsand vectors taught by Walker et al. as discussed herein. Briefly, Walkeret al., Journal of Applied Phycology, Vol. 17 (2005), pp. 363-368,reported stable nuclear transformation of Dunaliella tertiolecta withplasmid DNA. Using the transformation method of electroporation, Walkerintroduced the plasmid pDbleFLAG1.2 into Dunaliella tertiolecta.pDbleFLAG1.2 comprised sequence encoding a bleomycin antibioticresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotic phleomycin, operably linked to the promoter and 3′ UTR of theDunaliella tertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenasesmall subunit gene (rbcS1, GenBank Accession No. AY530155). Prior totransformation, Dunaliella tertiolecta was unable to be propagated inculture medium comprising 1 mg/L phleomycin. Upon transformation withthe pDbleFLAG1.2 plasmid, transformants of Dunaliella tertiolecta wereobtained that were propagated in selective culture medium comprising 1mg/L phleomycin. The expression of the ble gene product in Dunaliellatertiolecta enabled propagation in the presence of 1 mg/L phleomycin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Dunaliella tertiolecta.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. As reported by Walker, selection and maintenanceof transformed Dunaliella tertiolecta was conducted in Dunaliella medium(DM, as described by Provasoli et al., Archlv fur Mikrobiologie, Vol. 25(1957), pp. 392-428) further comprising 4.5 g/L NaCl and 1 mg/Lpheomycin. Additional media suitable for culturing Dunaliellatertiolecta for lipid production are discussed in Takagi et al., Journalof Bioscience and Bioengineering, Vol. 101:3 (2006), pp. 223-226 and inMassart and Hanston, Proceedings Venice 2010, Third InternationalSymposium on Energy from Biomass and Waste. Walker reported that theplasmid pDbleFLAG1.2 and the promoter and 3′ UTR of the Dunaliellatertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenase smallsubunit gene are suitable to enable heterologous expression inDunaliella tertiolecta. In addition, Walker reported that the bleomycinresistance cassette encoded on pDbleFLAG1.2 was suitable for use as aselectable marker in Dunaliella tertiolecta.

In an embodiment of the present invention, vector pDbleFLAG1.2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Dunaliella tertiolecta to reflect thecodon bias inherent in nuclear genes of Dunaliella tertiolecta inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the rbcS1 promoter upstream of the protein-codingsequence and operably linked to the rbcS1 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella tertiolecta genome for targeted genomic integration ofthe transformation vector. Homology regions may be selected to disruptone or more genomic sites of endogenous lipid biosynthesis pathwaygenes. Stable transformation of Dunaliella tertiolecta with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product may be used as a marker to select for Dunaliellatertiolecta transformed with the transformation vector on, but notlimited to, DM medium comprising pheomycin. Growth medium suitable forDunaliella tertiolecta lipid production include, but are not limited toDM medium and those culture media described by Takagi et al. and Massartand Hanston. Evaluation of fatty acid profiles of Dunaliella tertiolectalipids may be assessed through standard lipid extraction and analyticalmethods described herein.

Example 15 Engineering Volvox carteri

Expression of recombinant genes in accordance with the present inventionin Volvox carteri may be accomplished by modifying the methods andvectors taught by Hallman and Rappel et al. as discussed herein.Briefly, Hallman and Rappel et al., The Plant Journal, Volume 17 (1999),pp. 99-109, reported the stable nuclear transformation of Volvox carteriwith plasmid DNA. Using the transformation method of microprojectilebombardment, Hallman and Rappel introduced the pzeoE plasmid into Volvoxcarteri. The pzeoE plasmid comprised sequence encoding a bleomycinantibiotic resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein (ble), forresistance to the antibiotic zeocin, operably linked to and the promoterand 3′ UTR of the Volvox carteri beta-tubulin gene (GenBank AccessionNo. L24547). Prior to transformation, Volvox carteri was unable to bepropagated in culture medium comprising 1.5 ug/ml zeocin. Upontransformation with the pzeoE plasmid, transformants of Volvox carteriwere obtained that were propagated in selective culture mediumcomprising greater than 20 ug/ml zeocin. The expression of the ble geneproduct in Volvox carteri enabled propagation in the presence of 20ug/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use in Volvoxcarteri. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. As reported by Hallman and Rappel,selection and maintenance of transformed Volvox carteri was conducted inVolvox medium (VM, as described by Provasoli and Pintner, The Ecology ofAlgae, Special Publication No. 2 (1959), Tyron, C. A. and Hartman, R.T., eds., Pittsburgh: Univeristy of Pittsburgh, pp. 88-96) with 1 mg/Lpheomycin. Media suitable for culturing Volvox carteri for lipidproduction are also discussed by Starr in Starr R. C., Dev Biol Suppl.,Vol. 4 (1970), pp. 59-100). Hallman and Rappel reported that the plasmidpzeoE and the promoter and 3′ UTR of the Volvox carteri beta-tubulingene are suitable to enable heterologous expression in Volvox carteri.In addition, Hallman and Rappel reported that the bleomycin resistancecassette encoded on pzeoE was suitable for use as a selectable marker inVolvox carteri. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inVolvox carteri and suitable for use as selective markers Volvox carteriin have been reported (for instance see Hallamann and Sumper,Proceedings of the National Academy of Sciences, Vol. 91 (1994), pp11562-11566 and Hallman and Wodniok, Plant Cell Reports, Volume 25(2006), pp. 582-581).

In an embodiment of the present invention, vector pzeoE, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Volvox carteri to reflect the codonbias inherent in nuclear genes of Volvox carteri in accordance withTables 24A-D. For each lipid biosynthesis pathway protein of Table 25,the codon-optimized gene sequence can individually be operably linked tothe Volvox carteri beta-tubulin promoter upstream of the protein-codingsequence and operably linked to the Volvox carteri beta-tubulin3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Volvox carteri genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Volvox carteri genome(referenced in the publication by Prochnik et al., Science, Vol.329:5988 (2010), pp 223-226). Stable transformation of Volvox carteriwith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the ble gene product may be used as a markerto select for Volvox carteri transformed with the transformation vectoron, but not limited to, VM medium comprising zeocin. Growth mediumsuitable for Volvox carteri lipid production include, but are notlimited to VM medium and those culture media discussed by Starr.Evaluation of fatty acid profiles of Volvox carteri lipids may beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 16 Engineering Haematococcus pluvialis

Expression of recombinant genes in accordance with the present inventionin Haematococcus pluvialis may be accomplished by modifying the methodsand vectors taught by Steinbrenner and Sandmann et al. as discussedherein. Briefly, Steinbrenner and Sandmann et al., Applied andEnvironmental Microbiology, Vol. 72:12 (2006), pp. 7477-7484, reportedthe stable nuclear transformation of Haematococcus pluvialis withplasmid DNA. Using the transformation method of microprojectilebombardment, Steinbrenner introduced the plasmid pPlat-pds-L504R intoHaematococcus pluvialis. The plasmid pPlat-pds-L504R comprised anorflurazon resistance cassette, which comprised the promoter,protein-coding sequence, and 3′UTR of the Haematococcus pluvialisphytoene desaturase gene (Pds, GenBank Accession No. AY781170), whereinthe protein-coding sequence of Pds was modified at position 504 (therebychanging a leucine to an arginine) to encode a gene product (Pds-L504R)that confers resistance to the herbicide norflurazon. Prior totransformation with pPlat-pds-L504R, Haematococcus pluvialis was unableto propagate on medium comprising 5 uM norflurazon. Upon transformationwith the pPlat-pds-L504R plasmid, transformants of Haematococcuspluvialis were obtained that were propagated in selective culture mediumcomprising 5 uM norflurazon. The expression of the Pds-L504R geneproduct in Haematococcus pluvialis enabled propagation in the presenceof 5 uM norflurazon, thereby establishing the utility of the norflurazonherbicide resistance cassette as selectable marker for use inHaematococcus pluvialis. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. As reported bySteinbrenner, selection and maintenance of transformed Haematococcuspluvialis was conducted on agar plates comprising OHA medium (OHM (0.41g/L KNO₃, 0.03 g/L Na₂HPO₄, 0.246 g/L MgSO₄.7H₂O, 0.11 g/L CaCl₂.2H₂O,2.62 mg/L Fe_((III))citrate×H₂O, 0.011 mg/L CoCl₂.6H₂O, 0.012 mg/LCuSO₄.5H₂O, 0.075 mg/L Cr₂O₃, 0.98 mg/L MnCl₂.4H₂O, 0.12 mg/LNa₂MoO_(4∴2)H₂O, 0.005 mg/L SeO₂ and 25 mg/L biotin, 17.5 mg/L thiamine,and 15 mg/L vitamin B12), supplemented with 2.42 g/L Tris-acetate, and 5mM norflurazon. Propagation of Haematococcus pluvialis in liquid culturewas performed by Steinbrenner and Sandmann using basal medium (basalmedium as described by Kobayashi et al., Applied and EnvironmentalMicrobiology, Vol. 59 (1993), pp. 867-873). Steinbrenner and Sandmannreported that the pPlat-pds-L504R plasmid and promoter and 3′ UTR of theHaematococcus pluvialis phytoene desaturase gene are suitable to enableheterologous expression in Haematococcus pluvialis. In addition,Steinbrenner and Sandmann reported that the norflurazon resistancecassette encoded on pPlat-pds-L504R was suitable for use as a selectablemarker in Haematococcus pluvialis. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Haematococcus pluvialis have beenreported (see Kathiresan et al., Journal of Phycology, Vol. 45 (2009),pp 642-649).

In an embodiment of the present invention, vector pPlat-pds-L504R,comprising the nucleotide sequence encoding the Pds-L504R gene productfor use as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Haematococcus pluvialis to reflect thecodon bias inherent in nuclear genes of Haematococcus pluvialis inaccordance with Tables 24 A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Haematococcus pluvialis pds gene promoterupstream of the protein-coding sequence and operably linked to theHaematococcus pluvialis pds gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Haematococcuspluvialis genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Haematococcus pluvialis with the transformation vectoris achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thePds-L504R gene product may be used as a marker to select forHaematococcus pluvialis transformed with the transformation vector on,but not limited to, OHA medium comprising norflurazon. Growth mediasuitable for Haematococcus pluvialis lipid production include, but arenot limited to basal medium and those culture media described byKobayashi et al., Kathiresan et al, and Gong and Chen, Journal ofApplied Phycology, Vol. 9:5 (1997), pp. 437-444). Evaluation of fattyacid profiles of Haematococcus pluvialis lipids may be assessed throughstandard lipid extraction and analytical methods described herein.

Example 17 Engineering Closterium peracerosum-strigosum-littoralecomplex

Expression of recombinant genes in accordance with the present inventionin Closterium peracerosum-strigosum-littorals complex may beaccomplished by modifying the methods and vectors taught by Abe et al.as discussed herein. Briefly, Abe et al., Plant Cell Physiology, Vol.52:9 (2011), pp. 1676-1685, reported the stable nuclear transformationof Closterium peracerosum-strigosum-littorals complex with plasmid DNA.Using the transformation methods of microprojectile bombardment, Abeintroduced the plasmid pSA106 into Closteriumperacerosum-strigosum-littorals complex. Plasmid pSA106 comprised ableomycin resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein gene (ble,GenBank Accession No. CAA37050) operably linked to the promoter and 3′UTR of the Closterium peracerosum-strigosum-littorale complexChlorophyll a/b-binding protein gene (CAB, GenBank Accession No.AB363403). Prior to transformation with pSA106, Closteriumperacerosum-strigosum-littorale complex was unable to propagate onmedium comprising 3 ug/ml phleomycin. Upon transformation with pSA106,transformants of Closterium peracerosum-strigosum-littorale complex wereobtained that were propagated in selective culture medium comprising 3ug/ml phleomycin. The expression of the ble gene product in Closteriumperacerosum-strigosum-littorals complex enabled propagation in thepresence of 3 ug/ml phleomycin, thereby establishing the utility of thebleomycin antibiotic resistance cassette as selectable marker for use inClosterium peracerosum-strigosum-littorals complex. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. As reported by Abe, selection and maintenance of transformedClosterium peracerosum-strigosum-littorals complex was conducted firstin top agar with C medium (0.1 g/L KNO₃, 0.015 g/L Ca(NO₃)₂.4H2O, 0.05g/L glycerophosphate-Na2, 0.04 g/L MgSO₄.7H₂O, 0.5 g/L Tris(hydroxylmethyl) aminomethane, trace minerals, biotin, vitamins B₁ andB₁₂) and then subsequently isolated to agar plates comprising C mediumsupplemented with phleomycin. As reported by Abe, propagation ofClosterium peracerosum-strigosum-littorale complex in liquid culture wasperformed in C medium. Additional liquid culture medium suitable forpropagation of Closterium peracerosum-strigosum-littorals complex arediscussed by Sekimoto et al., DNA Research, 10:4 (2003), pp. 147-153.Abe reported that the pSA106 plasmid and promoter and 3′ UTR of theClosterium peracerosum-strigosum-littorale complex CAB gene are suitableto enable heterologous gene expression in Closteriumperacerosum-strigosum-littorals complex. In addition, Abe reported thatthe bleomycin resistance cassette encoded on pSA106 was suitable for useas a selectable marker in Closterium peracerosum-strigosum-littoralscomplex. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inClosterium peracerosum-strigosum-littorals complex have been reported(see Abe et al., Plant Cell Physiology, Vol. 49 (2008), pp. 625-632).

In an embodiment of the present invention, vector pSA106, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Closteriumperacerosum-strigosum-littorals complex to reflect the codon biasinherent in nuclear genes of Closterium peracerosum-strigosum-littoralscomplex in accordance with Tables 24A-D. For each lipid biosynthesispathway protein of Table 25, the codon-optimized gene sequence canindividually be operably linked to the Closteriumperacerosum-strigosum-littorals complex CAB gene promoter upstream ofthe protein-coding sequence and operably linked to the Closteriumperacerosum-strigosum-littorals complex CAB gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Closterium peracerosum-strigosum-littorals complex genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofClosterium peracerosum-strigosum-littorale complex with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the ble gene product may be used as a marker to select forClosterium peracerosum-strigosum-littorals complex transformed with thetransformation vector on, but not limited to, C medium comprisingphleomycin. Growth media suitable for Closteriumperacerosum-strigosum-littorals complex lipid production include, butare not limited to C medium and those culture media reported by Abe etal. and Sekimoto et al. Evaluation of fatty acid profiles of Closteriumperacerosum-strigosum-littorals complex lipids may be assessed throughstandard lipid extraction and analytical methods described herein.

Example 18 Engineering Dunaliella viridis

Expression of recombinant genes in accordance with the present inventionin Dunaliella viridis may be accomplished by modifying the methods andvectors taught by Sun et al. as discussed herein. Briefly, Sun et al.,Gene, Vol. 377 (2006), pp.140-149, reported the stable transformation ofDunaliella viridis with plasmid DNA. Using the transformation method ofelectoporation, Sun introduced the plasmid pDVNR, encoding the fullDunaliella viridis nitrate reductase gene into mutant Dunaliella viridis(Dunaliella viridis NR-mutants.) The NR-mutants are incapable of growthwithout the use of nitrate as a source of nitrogen. Nitrate reductasecatalyzes the conversion of nitrate to nitrite. Prior to transformation,Dunaliella viridis NR-mutants were unable to propagate in culture mediumcomprising nitrate (NO₃ ⁻) as the sole nitrogen source. The expressionof the Dunaliella viridis NR gene product in NR-mutant Dunaliellaviridis was used as a selectable marker to rescue the nitrate metabolismdeficiency. Upon transformation with the pDVNR plasmid, NR-mutantDunaliella viridis stably expressing the Dunaliella viridis NR geneproduct were obtained that were able to grow on agar plates comprisingnitrate as the sole carbon source. Evaluation of the DNA of the stabletransformants was performed by Southern analysis. Selection andmaintenance of the transformed Dunaliella viridis (NR mutant) wasperformed on agar plates comprising 5 mM KNO₃. Sun also reported thepropagation of Dunaliella viridis and Dunaliella viridis NR mutants inliquid culture medium. Additional media suitable for propagation ofDunaliella viridis are reported by Gordillo et al., Journal of AppliedPhycology, Vol. 10:2 (1998), pp. 135-144 and by Moulton and Burford,Hydrobiologia, Vols. 204-205:1 (1990), pp. 401-408. Sun reported thatthe plasmid pDVNR and the promoter and 3′ UTR/terminator of theDunaliella viridis nitrate reductase gene were suitable to enableheterologous expression in Dunaliella viridis NR-mutants. Sun alsoreported that expression of the Dunaliella viridis nitrate reductasegene product was suitable for use as a selectable marker in Dunaliellaviridis NR-mutants.

In an embodiment of the present invention, vector pDVNR, comprising thenucleotide sequence encoding the Dunaliella viridis nitrate reductase(DvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected Table 25, each protein-codingsequence codon-optimized for expression in Dunaliella viridis to reflectthe codon bias inherent in nuclear genes of Dunaliella viridis inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the DvNR promoter upstream of the protein-codingsequence and operably linked to the DvNR 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella viridis genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Dunaliella viridis NR mutants with thetransformation vector is achieved through well-known transformationtechniques including electorporation or other known methods. Activity ofthe DvNR gene product may be used as a selectable marker to rescue thenitrogen assimiliation deficiency of Dunaliella viridis NR mutantstrains and to select for Dunaliella viridis NR-mutants stablyexpressing the transformation vector. Growth media suitable forDunaliella viridis lipid production include, but are not limited tothose discussed by Sun et al., Moulton and Burford, and Gordillo et al.Evaluation of fatty acid profiles of Dunaliella viridis lipids may beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 19 Engineering Dunaliella Salina

Expression of recombinant genes in accordance with the present inventionin Dunaliella salina may be accomplished by modifying the methods andvectors taught by Geng et al. as discussed herein. Briefly, Geng et al.,Journal of Applied Phycology, Vol. 15 (2003), pp. 451-456, reported thestable transformation of Dunaliella salina with plasmid DNA. Using thetransformation method of electroporation, Geng introduced thepUΩHBsAg-CAT plasmid into Dunaliella salina. pUΩHBsAg-CAT comprises ahepatitis B surface antigen (HBsAG) expression cassette comprisingsequence encoding the hepatitis B surface antigen operably linked to aZea mays ubil promoter upstream of the HBsAG protein-coding region andoperably linked to the 3′UTR/terminator of the Agrobacterium tumefaciensnopaline synthase gene (nos) downstream of the HBsAG protein-codingregion. pUΩHBsAg-CAT further comprised a chloramphenicol resistancecassette, comprising sequence encoding the chloramphenicolacetyltransferase (CAT) gene product, conferring resistance to theantibiotic chloramphenicol, operably linked to the simian virus 40promoter and enhancer. Prior to transformation with pUΩHBsAg-CAT,Dunaliella salina was unable to propagate on medium comprising 60 mg/Lchloramphenicol. Upon transformation with the pUΩHBsAg-CAT plasmid,transformants of Dunaliella salina were obtained that were propagated inselective culture medium comprising 60 mg/L chloramphenicol. Theexpression of the CAT gene product in Dunaliella salina enabledpropagation in the presence of 60 mg/L chloramphenicol, therebyestablishing the utility of the chloramphenicol resistance cassette asselectable marker for use in Dunaliella salina. Detectable activity ofthe HBsAg gene product indicated that ubil promoter and nos3′UTR/terminator are suitable for enabling gene expression in Dunaliellasalina. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. Geng reported that selection andmaintenance of the transformed Dunaliella salina was performed on agarplates comprising Johnson's medium (J1, described by Borowitzka andBorowitzka (eds), Micro-algal Biotechnology. Cambridge University Press,Cambridge, pp. 460-461) with 60 mg/L chloramphenicol. Liquid propagationof Dunaliella salina was performed by Geng in J1 medium with 60 mg/Lchloramphenicol. Propagation of Dunaliella salina in media other than J1medium has been discussed (see Feng et al., Mol. Bio. Reports, Vol. 36(2009), pp. 1433-1439 and Borowitzka et al., Hydrobiologia, Vols.116-117:1 (1984), pp. 115-121). Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Dunaliella salina have been reported byFeng et al. Geng reported that the plasmid pUΩHBsAg-CAT, the ubilpromoter, and the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator are suitable to enable exogenous gene expression inDunaliella salina. In addition, Geng reported that the CAT resistancecassette encoded on pUΩHBsAg-CAT was suitable for use as a selectablemarker in Dunaliella salina.

In an embodiment of the present invention, vector pUΩHBsAg-CAT,comprising the nucleotide sequence encoding the CAT gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 25, each protein-coding sequence codon-optimizedfor expression in Dunaliella salina to reflect the codon bias inherentin nuclear genes of Dunaliella salina in accordance with Tables 24A-D.For each lipid biosynthesis pathway protein of Table 25, thecodon-optimized gene sequence can individually be operably linked to theubi1 promoter upstream of the protein-coding sequence and operablylinked to the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Dunaliella salina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Dunaliella salinawith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the CAT gene product may be used as a selectablemarker to select for Dunaliella salina transformed with thetransformation vector in, but not limited to, J1 medium comprisingchrloramphenicol. Growth medium suitable for Dunaliella salina lipidproduction include, but are not limited to J1 medium and those culturemedia described by Feng et al. and Borowitzka et al. Evaluation of fattyacid profiles of Dunaliella salina lipids may be assessed throughstandard lipid extraction and analytical methods described herein.

Example 20 Engineering Gonium pectoral

Expression of recombinant genes in accordance with the present inventionin Gonium pectoral may be accomplished by modifying the methods andvectors taught by Lerche and Hallman et al. as discussed herein.Briefly, Lerche and Hallman et al., BMC Biotechnology, Volume 9:64,2009, reported the stable nuclear transformation of Gonium pectoralewith plasmid DNA. Using the transformation method of microprojectilebombardment, Lerche introduced the plasmid pPmr3 into Gonium pectorale.Plasmid pPmr3 comprised a paromomycin resistance cassette, comprising asequence encoding the aminoglycoside 3′-phosphotransferase (aphVIII)gene product (GenBank Accession No. AAB03856) of Streptomyces rimosusfor resistance to the antibiotic paromomycin, operably linked to theVolvox carteri hsp70A-rbcS3 hybrid promoter upstream of the aphVIIIprotein-coding region and operably linked to the 3′ UTR/terminator ofthe Volvox carteri rbcS3 gene downstream of the aphVIII protein-codingregion. Prior to transformation with pPmr3, Gonium pectorale was unableto propagate on medium comprising 0.06 ug/ml paromomycin. Upontransformation with pPmr3, transformants of Gonium pectorale wereobtained that were propagated in selective culture medium comprising0.75 and greater ug/ml paromomycin. The expression of the aphVIII geneproduct in Gonium pectorale enabled propagation in the presence of 0.75and greater ug/ml paromomycin, thereby establishing the utility of theparomomycin antibiotic resistance cassette as selectable marker for usein Gonium pectorale. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Lerche and Hallmanreported that selection and maintenance of the transformed Goniumpectorale was performed in liquid Jaworski's medium (20 mg/LCa(NO₃)₂.4H₂O, 12.4 mg/L KH₂PO₄, 50 mg/L MgSO₄.7H₂O, 15.9 mg/L NaHCO₃,2.25 mg/L EDTA-FeNa, 2.25 mg/L EDTA Na₂, 2.48 g/L H₃BO₃, 1.39 g/LMnCl₂.4H₂O, 1 mg/L (NH₄)₆MO₇O₂4.4H₂O, 0.04 mg/L vitamin B12, 0.04 mg/LThiamine-HCl, 0.04 mg/L biotin, 80 mg/L NaNO₃, 36 mg/L Na₄HPO₄.12H₂O)with 1.0 ug/ml paromomycin. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Gonium pectorale are further discussedby Lerche and Hallman. Lerche and Hallman reported that the plasmidpPmr3, Volvox carteri hsp70A-rbcS3 hybrid promoter, and the 3′UTR/terminator of the Volvox carteri rbcS3 gene are suitable to enableexogenous gene expression in Gonium pectorals. In addition, Lerche andHallman reported that the paromomycin resistance cassette encoded pPmr3was suitable for use as a selectable marker in Gonium pectorale.

In an embodiment of the present invention, vector pPmr3, comprising thenucleotide sequence encoding the aphVIII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 25, each protein-coding sequence codon-optimizedfor expression in Gonium pectorale to reflect the codon bias inherent innuclear genes of Gonium pectorale in accordance with Tables 24A-D. Foreach lipid biosynthesis pathway protein of Table 25, the codon-optimizedgene sequence can individually be operably linked to the Volvox carterihsp70A-rbcS3 hybrid promoter upstream of the protein-coding sequence andoperably linked to the Volvox carteri rbcS3 gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Gonium pectorale genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Gonium pectorale with the transformation vectormay be achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of theaphVIII gene product may be used as a selectable marker to select forGonium pectorale transformed with the transformation vector in, but notlimited to, Jaworski's medium comprising paromomycin. Growth mediasuitable for Gonium pectorale lipid production include Jawaorski'smedium and media reported by Stein, American Journal of Botany, Vol.45:9 (1958), pp. 664-672. Evaluation of fatty acid profiles of Goniumpectorale lipids may be assessed through standard lipid extraction andanalytical methods described herein.

Example 21 Engineering Phaeodactylum tricornutum

Expression of recombinant genes in accordance with the present inventionin Phaeodactylum tricornutum may be accomplished by modifying themethods and vectors taught by Apt et al. as discussed herein. Briefly,Apt et al., Molecular and General Genetics, Vol. 252 (1996), pp.572-579, reported the stable nuclear transformation of Phaeodactylumtricornutum with vector DNA. Using the transformation technique ofmicroproj ectile bombardment, Apt introduced the plasmid pfcpA intoPhaeodactylum tricornutum. Plasmid pfcpA comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics phleomycin and zeocin, operably linked to the promoter ofthe Phaeodactylum tricornutum fucoxanthin chlorophyll a binding proteingene (fcpA) upstream of the ble protein-coding region and operablylinked to the 3′ UTR/terminator of the Phaeodactylum tricornutum fcpAgene at the 3′ region, or downstream of the ble protein-coding region.Prior to transformation with pfcpA, Phaeodactylum tricornutum was unableto propagate on medium comprising 50 ug/ml zeocin. Upon transformationwith pfcpA, transformants of Phaeodactylum tricornutum were obtainedthat were propagated in selective culture medium comprising 50 ug/mlzeocin. The expression of the ble gene product in Phaeodactylumtricornutum enabled propagation in the presence of 50 ug/ml zeocin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Phaeodactylum tricornutum.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Apt reported that selection and maintenance of thetransformed Phaeodactylum tricornutum was performed on agar platescomprising LDM medium (as reported by Starr and Zeikus, Journal ofPhycology, Vol. 29, Supplement, (1993)) with 50 mg/L zeocin. Aptreported liquid propagation of Phaeodactylum tricornutum transformantsin LDM medium with 50 mg/L zeocin. Propagation of Phaeodactylumtricornutum in medium other than LDM medium has been discussed (byZaslayskaia et al., Science, Vol. 292 (2001), pp. 2073-2075, and byRadokovits et al., Metabolic Engineering, Vol. 13 (2011), pp. 89-95).Additional plasmids, promoters, 3′UTR/terminators, and selectablemarkers suitable for enabling heterologous gene expression inPhaeodactylum tricornutum have been reported in the same report by Aptet al., by Zaslayskaia et al., and by Radokovits et al.). Apt reportedthat the plasmid pfcpA, and the Phaeodactylum tricornutum fcpA promoterand 3′ UTR/terminator are suitable to enable exogenous gene expressionin Phaeodactylum tricornutum. In addition, Apt reported that thebleomycin resistance cassette encoded on pfcpA was suitable for use as aselectable marker in Phaeodactylum tricornutum.

In an embodiment of the present invention, vector pfcpA, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 25, each protein-coding sequence codon-optimizedfor expression in Phaeodactylum tricornutum to reflect the codon biasinherent in nuclear genes of Phaeodactylum tricornutum in accordancewith Tables 24A-D. For each lipid biosynthesis pathway protein of Table25, the codon-optimized gene sequence can individually be operablylinked to the Phaeodactylum tricornutum fcpA gene promoter upstream ofthe protein-coding sequence and operably linked to the Phaeodactylumtricornutum fcpA gene 3′UTR/terminator at the 3′ region, or downstream,of the protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Phaeodactylum tricornutumgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Phaeodactylumtricornutum genome (referenced in the publication by Bowler et al.,Nature, Vol. 456 (2008), pp. 239-244). Stable transformation ofPhaeodactylum tricornutum with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product maybe used as a marker to select for Phaeodactylum tricornutum transformedwith the transformation vector in, but not limited to, LDM mediumcomprising paromomycin. Growth medium suitable for Phaeodactylumtricornutum lipid production include, but are not limited to f/2 mediumas reported by Radokovits et al. Evaluation of fatty acid profiles ofPhaeodactylum tricornutum lipids may be assessed through standard lipidextraction and analytical methods described herein.

Example 22 Engineering Chaetoceros Sp

Expression of recombinant genes in accordance with the present inventionin Chaetoceros sp. may be accomplished by modifying the methods andvectors taught by Yamaguchi et al. as discussed herein. Briefly,Yamaguchi et al., Phycological Research, Vol. 59:2 (2011), pp. 113-119,reported the stable nuclear transformation of Chaetoceros sp. withplasmid DNA. Using the transformation method of microprojectilebombardment, Yamaguchi introduced the plasmid pTpfcp/nat intoChaetoceros sp. pTpfcp/nat comprised a nourseothricin resistancecassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/ terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Chaetoceros sp. was unable to propagate on mediumcomprising 500 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Chaetoceros sp. were obtained that werepropagated in selective culture medium comprising 500 ug/mlnourseothricin. The expression of the nat gene product in Chaetocerossp. enabled propagation in the presence of 500 ug/ml nourseothricin,thereby establishing the utility of the nourseothricin antibioticresistance cassette as selectable marker for use in Chaetoceros sp.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Yamaguchi reported that selection and maintenanceof the transformed Chaetoceros sp. was performed on agar platescomprising f/2 medium (as reported by Guilard, R. R., Culture ofPhytoplankton for feeding marine invertebrates, In Culture of MarineInvertebrate Animals, Smith and Chanley (eds) 1975, Plenum Press, NewYork, pp. 26-60) with 500 ug/ml nourseothricin. Liquid propagation ofChaetoceros sp. transformants, as performed by Yamaguchi, was carriedout in f/2 medium with 500 mg/L nourseothricin. Propagation ofChaetoceros sp. in additional culture medium has been reported (forexample in Napolitano et al., Journal of the World Aquaculture Society,Vol. 21:2 (1990), pp. 122-130, and by Volkman et al., Journal ofExperimental Marine Biology and Ecology, Vol. 128:3 (1989), pp.219-240). Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inChaetoceros sp. have been reported in the same report by Yamaguchi etal. Yamaguchi reported that the plasmid pTpfcp/nat, and theThalassiosira pseudonana fcp promoter and 3′ UTR/terminator are suitableto enable exogenous gene expression in Chaetoceros sp. In addition,Yamaguchi reported that the nourseothricin resistance cassette encodedon pTpfcp/nat was suitable for use as a selectable marker in Chaetocerossp.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in the closely-related Chaetoceroscompressum to reflect the codon bias inherent in nuclear genes ofChaetoceros compressum in accordance with Tables 24A-D. For each lipidbiosynthesis pathway protein of Table 25, the codon-optimized genesequence can individually be operably linked to the Thalassiosirapseudonana fcp gene promoter upstream of the protein-coding sequence andoperably linked to the Thalassiosira pseudonana fcp gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chaetoceros sp. genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chaetoceros sp.with the transformation vector is achieved through well-knowntransformation including microprojectile bombardment or other knownmethods. Activity of the nat gene product may be used as a selectablemarker to select for Chaetoceros sp. transformed with the transformationvector in, but not limited to, f/2 agar medium comprisingnourseothricin. Growth medium suitable for Chaetoceros sp. lipidproduction include, but are not limited to, f/2 medium, and thoseculture media discussed by Napolitano et al. and Volkman et al.Evaluation of fatty acid profiles of Chaetoceros sp lipids may beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 23 Engineering Cylindrotheca fusiformis

Expression of recombinant genes in accordance with the present inventionin Cylindrotheca fusiformis may be accomplished by modifying the methodsand vectors taught by Poulsen and Kroger et al. as discussed herein.Briefly, Poulsen and Kroger et al., FEBS Journal, Vol. 272 (2005), pp.3413-3423, reported the transformation of Cylindrotheca fusiformis withplasmid DNA. Using the transformation method of microproj ectilebombardment, Poulsen and Kroger introduced the pCF-ble plasmid intoCylindrotheca fusiformis. Plasmid pCF-ble comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics zeocin and phleomycin, operably linked to the Cylindrothecafusiformis fucozanthin chlorophyll a/c binding protein gene (fcpA,GenBank Accesssion No. AY125580) promoter upstream of the bleprotein-coding region and operably linked to the Cylindrothecafusiformis fcpA gene 3′UTR/terminator at the 3′ region (down-stream ofthe ble protein-coding region). Prior to transformation with pCF-ble,Cylindrotheca fusiformis was unable to propagate on medium comprising 1mg/ml zeocin. Upon transformation with pCF-ble, transformants ofCylindrotheca fusiformis were obtained that were propagated in selectiveculture medium comprising 1 mg/ml zeocin. The expression of the ble geneproduct in Cylindrotheca fusiformis enabled propagation in the presenceof 1 mg/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use inCylindrotheca fusiformis. Poulsen and Kroger reported that selection andmaintenance of the transformed Cylindrotheca fusiformis was performed onagar plates comprising artificial seawater medium with 1 mg/ml zeocin.Poulsen and Kroger reported liquid propagation of Cylindrothecafusiformis transformants in artificial seawater medium with 1 mg/mlzeocin. Propagation of Cylindrotheca fusiformis in additional culturemedium has been discussed (for example in Liang et al., Journal ofApplied Phycology, Vol. 17:1 (2005), pp. 61-65, and by Orcutt andPatterson, Lipids, Vol. 9:12 (1974), pp. 1000-1003). Additionalplasmids, promoters, and 3′UTR/terminators for enabling heterologousgene expression in Chaetoceros sp. have been reported in the same reportby Poulsen and Kroger. Poulsen and Kroger reported that the plasmidpCF-ble and the Cylindrotheca fusiformis fcp promoter and 3′UTR/terminator are suitable to enable exogenous gene expression inCylindrotheca fusiformis. In addition, Poulsen and Kroger reported thatthe bleomycin resistance cassette encoded on pCF-ble was suitable foruse as a selectable marker in Cylindrotheca fusiformis.

In an embodiment of the present invention, vector pCF-ble, comprisingthe nucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 25, each protein-coding sequence codon-optimizedfor expression in Cylindrotheca fusiformis to reflect the codon biasinherent in nuclear genes of Cylindrotheca fusiformis in accordance withTables 24A-D. For each lipid biosynthesis pathway protein of Table 25,the codon-optimized gene sequence can individually be operably linked tothe Cylindrotheca fusiformis fcp gene promoter upstream of theprotein-coding sequence and operably linked to the Cylindrothecafusiformis fcp gene 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Cylindrotheca fusiformisgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofCylindrotheca fusiformis with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product maybe used as a selectable marker to select for Cylindrotheca fusiformistransformed with the transformation vector in, but not limited to,artificial seawater agar medium comprising zeocin. Growth media suitablefor Cylindrotheca fusiformis lipid production include, but are notlimited to, artificial seawater and those media reported by Liang et al.and Orcutt and Patterson. Evaluation of fatty acid profiles ofCylindrotheca fusiformis lipids may be assessed through standard lipidextraction and analytical methods described herein.

Example 24 Engineering Amphidinium Sp

Expression of recombinant genes in accordance with the present inventionin Amphidinium sp. may be accomplished by modifying the methods andvectors taught by ten Lohuis and Miller et al. as discussed herein.Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol. 13:3(1998), pp. 427-435, reported the stable transformation of Amphidiniumsp. with plasmid DNA. Using the transformation technique of agitation inthe presence of silicon carbide whiskers, ten Lohuis introduced theplasmid pMT NPT/GUS into Amphidinium sp. pMT NPT/GUS comprised aneomycin resistance cassette, comprising sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) operably linked to the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter upstream, or 5′ of the nptII protein-codingregion and operably linked to the 3′ UTR/terminator of the nos gene atthe 3′ region (down-stream of the nptII protein-coding region). ThenptII gene product confers resistance to the antibiotic G418. The pMTNPT/GUS plasmid further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably-linked to a CaMV 35S promoter andfurther operably linked to the CaMV 35S 3′ UTR/terminator. Prior totransformation with pMT NPT/GUS, Amphidinium sp. was unable to bepropagated on medium comprising 3 mg/ml G418. Upon transformation withpMT NPT/GUS, transformants of Amphidinium sp. were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Amphidinium sp. enabledpropagation in the presence of 3 mg/ml G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Amphidinium sp. Detectable activity of the GUSreporter gene indicated that CaMV 35S promoter and 3′UTR are suitablefor enabling gene expression in Amphidinium sp. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. ten Lohuis and Miller reported liquid propagation ofAmphidinium sp transformants in medium comprising seawater supplementedwith F/2 enrichment solution (provided by the supplier Sigma) and 3mg/ml G418 as well as selection and maintenance of Amphidinium sp.transformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Amphidinium sp. inadditional culture medium has been reported (for example in Mansour etal., Journal of Applied Phycology, Vol. 17:4 (2005) pp. 287-v300). Anadditional plasmid, comprising additional promoters, 3′UTR/terminators,and a selectable marker for enabling heterologous gene expression inAmphidinium sp. have been reported in the same report by ten Lohuis andMiller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUS andthe promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Amphidinium sp. Inaddition, ten Lohuis and Miller reported that the neomycin resistancecassette encoded on pMT NPT/GUS was suitable for use as a selectablemarker in Amphidinium sp.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Amphidinium sp. to reflect the codonbias inherent in nuclear genes of the closely-related species,Amphidinium carterae in accordance with Tables 24A-D. For each lipidbiosynthesis pathway protein of Table 25, the codon-optimized genesequence can individually be operably linked to the Agrobacteriumtumefaciens nopaline synthase (nos) gene promoter upstream of theprotein-coding sequence and operably linked to the nos 3′UTR/terminatorat the 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Amphidinium sp. genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Amphidinium sp. with the transformation vectoris achieved through well-known transformation techniques includingsilicon fibre-mediated microinjection or other known methods. Activityof the nptII gene product may be used as a selectable marker to selectfor Amphidinium sp. transformed with the transformation vector in, butnot limited to, seawater agar medium comprising G418. Growth mediasuitable for Amphidinium sp. lipid production include, but are notlimited to, artificial seawater and those media reported by Mansour etal. and ten Lohuis and Miller. Evaluation of fatty acid profiles ofAmphidinium sp. lipids may be assessed through standard lipid extractionand analytical methods described herein.

Example 25 Engineering Symbiodinium microadriacticum

Expression of recombinant genes in accordance with the present inventionin Symbiodinium microadriacticum may be accomplished by modifying themethods and vectors taught by ten Lohuis and Miller et al. as discussedherein. Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol.13:3 (1998), pp. 427-435, reported the stable transformation ofSymbiodinium microadriacticum with plasmid DNA. Using the transformationtechnique of silicon fibre-mediated microinjection, ten Lohuisintroduced the plasmid pMT NPT/GUS into Symbiodinium microadriacticum.pMT NPT/GUS comprised a neomycin resistance cassette, comprisingsequence encoding the neomycin phosphotransferase II (nptII) geneproduct (GenBank Accession No. AAL92039) operably linked to theAgrobacterium tumefaciens nopaline synthase (nos) gene promoterupstream, or 5′ of the nptII protein-coding region and operably linkedto the 3′ UTR/terminator of the nos gene at the 3′ region (down-streamof the nptII protein-coding region). The nptII gene product confersresistance to the antibiotic G418. The pMT NPT/GUS plasmid furthercomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter and further operablylinked to the CaMV 35S 3′ UTR/terminator. Prior to transformation withpMT NPT/GUS, Symbiodinium microadriacticum was unable to be propagatedon medium comprising 3 mg/ml G418. Upon transformation with pMT NPT/GUS,transformants of Symbiodinium microadriacticum were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Symbiodinium microadriacticumenabled propagation in the presence of 3 mg/ml G418, therebyestablishing the utility of the neomycin antibiotic resistance cassetteas selectable marker for use in Symbiodinium microadriacticum.Detectable activity of the GUS reporter gene indicated that CaMV 35Spromoter and 3′UTR are suitable for enabling gene expression inSymbiodinium microadriacticum. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. ten Lohuis andMiller reported liquid propagation of Symbiodinium microadriacticumtransformants in medium comprising seawater supplemented with F/2enrichment solution (provided by the supplier Sigma) and 3 mg/ml G418 aswell as selection and maintenance of Symbiodinium microadriacticumtransformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Symbiodiniummicroadriacticum in additional culture medium has been discussed (forexample in Iglesias-Prieto et al., Proceedings of the National Academyof Sciences, Vol. 89:21 (1992) pp. 10302-10305). An additional plasmid,comprising additional promoters, 3′UTR/terminators, and a selectablemarker for enabling heterologous gene expression in Symbiodiniummicroadriacticum have been discussed in the same report by ten Lohuisand Miller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUSand the promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Symbiodiniummicroadriacticum. In addition, ten Lohuis and Miller reported that theneomycin resistance cassette encoded on pMT NPT/GUS was suitable for useas a selectable marker in Symbiodinium microadriacticum.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 25, each protein-coding sequence codon-optimizedfor expression in Symbiodinium microadriacticum to reflect the codonbias inherent in nuclear genes of Symbiodinium microadriacticum inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Agrobacterium tumefaciens nopaline synthase(nos) gene promoter upstream of the protein-coding sequence and operablylinked to the nos 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Symbiodiniummicroadriacticum genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Symbiodinium microadriacticum with thetransformation vector is achieved through well-known transformationtechniques including silicon fibre-mediated microinjection or otherknown methods. Activity of the nptII gene product may be used as aselectable marker to select for Symbiodinium microadriacticumtransformed with the transformation vector in, but not limited to,seawater agar medium comprising G418. Growth media suitable forSymbiodinium microadriacticum lipid production include, but are notlimited to, artificial seawater and those media reported byIglesias-Prieto et al. and ten Lohuis and Miller. Evaluation of fattyacid profiles of Symbiodinium microadriacticum lipids may be assessedthrough standard lipid extraction and analytical methods describedherein.

Example 26 Engineering Nannochloropsis Sp

Expression of recombinant genes in accordance with the present inventionin Nannochloropsis sp. W2J3B may be accomplished by modifying themethods and vectors taught by Kilian et al. as discussed herein.Briefly, Kilian et al., Proceedings of the National Academy of Sciences,Vol. 108:52 (2011) pp. 21265-21269, reported the stable nucleartransformation of Nannochloropsis with a transformation construct. Usingthe transformation method of electroporation, Kilian introduced thetransformation construct C2 into Nannochloropsis sp. W2J3B. The C2transformation construct comprised a bleomycin resistance cassette,comprising the coding sequence for the Streptoalloteichus hindustanusBleomycin binding protein (ble), for resistance to the antibioticsphleomycin and zeocin, operably linked to and the promoter of theNannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding proteingene VCP2 upstream of the ble protein-coding region and operably linkedto the 3′UTR/terminator of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding gene VCP1 downstream of the bleprotein-coding region. Prior to transformation with C2, Nannochloropsissp. W2J3B was unable to propagate on medium comprising 2 ug/ml zeocin.Upon transformation with C2, transformants of Nannochloropsis sp. W2J3Bwere obtained that were propagated in selective culture mediumcomprising 2 ug/ml zeocin. The expression of the ble gene product inNannochloropsis sp. W2J3B enabled propagation in the presence of 2 ug/mlzeocin, thereby establishing the utility of the bleomycin antibioticresistance cassette as selectable marker for use in Nannochloropsis.Evaluation of the genomic DNA of the stable transformants was performedby PCR. Kilian reported liquid propagation of Nannochloropsis sp. W2J3Btransformants in F/2 medium (reported by Guilard and Ryther, CanadianJournal of Microbiology, Vol. 8 (1962), pp. 229-239) comprising fivefoldlevels of trace metals, vitamins, and phosphate solution, and furthercomprising 2 ug/ml zeocin. Kilian also reported selection andmaintenance of Nannochloropsis sp. W2J3B transformants on agar F/2medium comprising artificial seawater 2 mg/ml zeocin. Propagation ofNannochloropsis in additional culture medium has been discussed (forexample in Chiu et al., Bioresour Technol., Vol. 100:2 (2009), pp.833-838 and Pal et al., Applied Microbiology and Biotechnology, Vol.90:4 (2011), pp. 1429-1441.). Additional transformation constructs,comprising additional promoters and 3′UTR/terminators for enablingheterologous gene expression in Nannochloropsis sp. W2J3B and selectablemarkers for selection of transformants have been described in the samereport by Kilian. Kilian reported that the transformation construct C2and the promoter of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding protein gene VCP2 and 3′UTR/terminator of the Nannochloropsis sp. W2J3B violaxanthin/chlorophylla-binding protein gene VCP1 are suitable to enable exogenous geneexpression in Nannochloropsis sp. W2J3B. In addition, Kilian reportedthat the bleomycin resistance cassette encoded on C2 was suitable foruse as a selectable marker in Nannochloropsis sp. W2J3B.

In an embodiment of the present invention, transformation construct C2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Nannochloropsis sp. W2J3B to reflectthe codon bias inherent in nuclear genes of Nannochloropsis sp. inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Nannochloropsis sp. W2J3B VCP2 gene promoterupstream of the protein-coding sequence and operably linked to theNannochloropsis sp. W2J3B VCP1 gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to theNannochloropsis sp. W2J3B genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Nannochloropsis sp. W2J3B with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product may be used as a selectable marker to select forNannochloropsis sp. W2J3B transformed with the transformation vector in,but not limited to, F/2 medium comprising zeocin. Growth media suitablefor Nannochloropsis sp. W2J3B lipid production include, but are notlimited to, F/2 medium and those media reported by Chiu et al. and Palet al. Evaluation of fatty acid profiles of Nannochloropsis sp. W2J3Blipids may be assessed through standard lipid extraction and analyticalmethods described herein.

Example 27 Engineering Cyclotella cryptica

Expression of recombinant genes in accordance with the present inventionin Cyclotella cryptica may be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Cyclotella cryptica with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Cyclotella cryptica. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Cyclotella cryptica was unable to propagate on 50%artificial seawater medium comprising 100 ug/ml G418. Upontransformation with pACCNPT5.1, transformants of Cyclotella crypticawere obtained that were propagated in selective 50% artificial seawatermedium comprising 100 ug/ml G418. The expression of the nptII geneproduct in Cyclotella cryptica enabled propagation in the presence of100 ug/ml G418, thereby establishing the utility of the neomycinantibiotic resistance cassette as selectable marker for use inCyclotella cryptica. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Dunahay reportedliquid propagation of Cyclotella cryptica in artificial seawater medium(ASW, as discussed by Brown, L., Phycologia, Vol. 21 (1982), pp.408-410) supplemented with 1.07 mM sodium silicate and with 100 ug/mlG418. Dunahay also reported selection and maintenance of Cyclotellacryptica transformants on agar plates comprising ASW medium with 100ug/ml G418. Propagation of Cyclotella cryptica in additional culturemedium has been discussed (for example in Sriharan et al., AppliedBiochemistry and Biotechnology, Vol. 28-29:1 (1991), pp. 317-326 andPahl et al., Journal of Bioscience and Bioengineering, Vol. 109:3(2010), pp. 235-239). Dunahay reported that the plasmid pACCNPT5.1 andthe promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase)gene are suitable to enable exogenous gene expression in Cyclotellacryptica. In addition, Dunahay reported that the neomycin resistancecassette encoded on pACCNPT5.1 was suitable for use as a selectablemarker in Cyclotella cryptica.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Cyclotella cryptica to reflect thecodon bias inherent in nuclear genes of Cyclotella cryptica inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Cyclotella cryptica ACCase promoter upstreamof the protein-coding sequence and operably linked to the Cyclotellacryptica ACCase 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Cyclotella cryptica genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Cyclotella crypticawith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the nptII gene product may be used as amarker to select for for Cyclotella cryptica transformed with thetransformation vector in, but not limited to, agar ASW medium comprisingG418. Growth media suitable for Cyclotella cryptica lipid productioninclude, but are not limited to, ASW medium and those media reported bySriharan et al., 1991 and Pahl et al. Evaluation of fatty acid profilesof Cyclotella cryptica lipids may be assessed through standard lipidextraction and analytical methods described herein.

Example 28 Engineering Navicula saprophila

Expression of recombinant genes in accordance with the present inventionin Navicula saprophila may be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Navicula saprophila with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Navicula saprophila. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Navicula saprophila was unable to propagate on artificialseawater medium comprising 100 ug/ml G418. Upon transformation withpACCNPT5.1, transformants of Navicula saprophila were obtained that werepropagated in selective artificial seawater medium comprising 100 ug/mlG418. The expression of the nptII gene product in Navicula saprophilaenabled propagation in the presence of G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Navicula saprophila. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Dunahayreported liquid propagation of Navicula saprophila in artificialseawater medium (ASW, as discussed by Brown, L., Phycologia, Vol. 21(1982), pp. 408-410) supplemented with 1.07 mM sodium silicate and with100 ug/ml G418. Dunahay also reported selection and maintenance ofNavicula saprophila transformants on agar plates comprising ASW mediumwith 100 ug/ml G418. Propagation of Navicula saprophila in additionalculture medium has been discussed (for example in Tadros and Johansen,Journal of Phycology, Vol. 24:4 (1988), pp. 445-452 and Sriharan et al.,Applied Biochemistry and Biotechnology, Vol. 20-21:1 (1989), pp.281-291). Dunahay reported that the plasmid pACCNPT5.1 and the promoterof the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene aresuitable to enable exogenous gene expression in Navicula saprophila. Inaddition, Dunahay reported that the neomycin resistance cassette encodedon pACCNPT5.1 was suitable for use as a selectable marker in Naviculasaprophila.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Navicula saprophila to reflect thecodon bias inherent in nuclear genes of the closely-related Naviculapelliculosa in accordance with Tables 24A-D. For each lipid biosynthesispathway protein of Table 25, the codon-optimized gene sequence canindividually be operably linked to the Cyclotella cryptica ACCase genepromoter upstream of the protein-coding sequence and operably linked tothe Cyclotella cryptica ACCase gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to the Naviculasaprophila genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Navicula saprophila with the transformation vector isachieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thenptII gene product may be used as a selectable marker to select forNavicula saprophila transformed with the transformation vector in, butnot limited to, agar ASW medium comprising G418. Growth media suitablefor Navicula saprophila lipid production include, but are not limitedto, ASW medium and those media reported by Sriharan et al. 1989 andTadros and Johansen. Evaluation of fatty acid profiles of Naviculasaprophila lipids may be assessed through standard lipid extraction andanalytical methods described herein.

Example 29 Engineering Thalassiosira pseudonana

Expression of recombinant genes in accordance with the present inventionin Thalassiosira pseudonana may be accomplished by modifying the methodsand vectors taught by Poulsen et al. as discussed herein. Briefly,Poulsen et al., Journal of Phycology, Vol. 42 (2006), pp. 1059-1065,reported the stable transformation of Thalassiosira pseudonana withplasmid DNA. Using the transformation method of microproj ectilebombardment, Poulsen introduced the plasmid pTpfcp/nat in toThalassiosira pseudonana. pTpfcp/nat comprised a nourseothricinresistance cassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/ terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Thalassiosira pseudonana was unable to propagate onmedium comprising 10 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Thalassiosira pseudonana were obtained thatwere propagated in selective culture medium comprising 100 ug/mlnourseothricin. The expression of the nat gene product in Thalassiosirapseudonana enabled propagation in the presence of 100 ug/mlnourseothricin, thereby establishing the utility of the nourseothricinantibiotic resistance cassette as selectable marker for use inThalassiosira pseudonana. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Poulsen reported thatselection and maintenance of the transformed Thalassiosira pseudonanawas performed in liquid culture comprising modified ESAW medium (asdiscussed by Harrison et al., Journal of Phycology, Vol. 16 (1980), pp.28-35) with 100 ug/ml nourseothricin. Propagation of Thalassiosirapseudonana in additional culture medium has been discussed (for examplein Volkman et al., Journal of Experimental Marine Biology and Ecology,Vol. 128:3 (1989), pp. 219-240). An additional plasmid, comprisingadditional selectable markers suitable for use in Thalassiosirapseudonana has been discussed in the same report by Poulsen. Poulsenreported that the plasmid pTpfcp/nat, and the Thalassiosira pseudonanafcp promoter and 3′ UTR/terminator are suitable to enable exogenous geneexpression in Thalassiosira pseudonana. In addition, Poulsen reportedthat the nourseothricin resistance cassette encoded on pTpfcp/nat wassuitable for use as a selectable marker in Thalassiosira pseudonana.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Thalassiosira pseudonana to reflectthe codon bias inherent in nuclear genes of Thalassiosira pseudonana inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Thalassiosira pseudonana fcp gene promoterupstream of the protein-coding sequence and operably linked to theThalassiosira pseudonana fcp gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Thalassiosirapseudonana genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. One skilled in theart can identify such homology regions within the sequence of theThalassiosira pseudonana genome (referenced in the publication byArmbrust et al., Science, Vol. 306: 5693 (2004): pp. 79-86). Stabletransformation of Thalassiosira pseudonana with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nat gene product may be used as a marker to select forThalassiosira pseudonana transformed with the transformation vector inbut not limited to, ESAW agar medium comprising nourseothricin. Growthmedia suitable for Thalassiosira pseudonana lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Volkman et al. and Harrison et al. Evaluation of fatty acid profilesof Thalassiosira pseudonana lipids may be assessed through standardlipid extraction and analytical methods described herein.

Example 30 Engineering Chlamydomonas reinhardth

Expression of recombinant genes in accordance with the present inventionin Chlamydomonas reinhardtii may be accomplished by modifying themethods and vectors taught by Cerutti et al. as discussed herein.Briefly, Cerutti et al., Genetics, Vol. 145:1 (1997), pp. 97-110,reported the stable nuclear transformation of Chlamydomonas reinhardtiiwith a transformation vector. Using the transformation method ofmicroproj ectile bombardment, Cerutti introduced transformationconstruct P[1030] into Chlamydomonas reinhardtii. Construct P[1030]comprised a spectinomycin resistance cassette, comprising sequenceencoding the aminoglucoside 3″-adenyltransferase (aadA) gene productoperably linked to the Chlamydomonas reinhardtiiribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene(RbcS2, GenBank Accession No. X04472) promoter upstream of the aadAprotein-coding region and operably linked to the Chlamydomonasreinhardtii RbcS2 gene 3′ UTR/ terminator at the 3′ region (downstreamof the aadA protein coding-sequence). The aadA gene product confersresistance to the antibiotic spectinomycin. Prior to transformation withP[1030], Chlamydomonas reinhardtii was unable to propagate on mediumcomprising 90 ug/ml spectinomycin. Upon transformation with P[1030],transformants of Chlamydomonas reinhardtii were obtained that werepropagated in selective culture medium comprising 90 ug/mlspectinomycin, thereby establishing the utility of the spectinomycinantibiotic resistance cassette as a selectable marker for use inChlamydomonas reinhardtii. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Cerutti reported thatselection and maintenance of the transformed Chlamydomonas reinhardtiiwas performed on agar plates comprising Tris-acetate-phosphate medium(TAP, as described by Harris, The Chlamydomonas Sourcebook, AcademicPress, San Diego, 1989) with 90 ug/ml spectinomycin. Ceruttiadditionally reported propagation of Chlamydomonas reinhardtii in TAPliquid culture with 90 ug/ml spectinomycin. Propagation of Chlamydomonasreinhardtii in alternative culture medium has been discussed (forexample in Dent et al., African Journal of Microbiology Research, Vol.5:3 (2011), pp. 260-270 and Yantao et al., Biotechnology andBioengineering, Vol. 107:2 (2010), pp. 258-268). Additional constructs,comprising additional selectable markers suitable for use inChlamydomonas reinhardtii as well as numerous regulatory sequences,including protomers and 3′ UTRs suitable for promoting heterologous geneexpression in Chlamydomonas reinhardtii are known in the art and havebeen discussed (for a review, see Radakovits et al., Eurkaryotic Cell,Vol. 9:4 (2010), pp. 486-501). Cerutti reported that the transformationvector P[1030] and the Chlamydomonas reinhardtii promoter and 3′UTR/terminator are suitable to enable exogenous gene expression inChlamydomonas reinhardtii. In addition, Cerutti reported that thespectinomycin resistance cassette encoded on P[1030] was suitable foruse as a selectable marker in Chlamydomonas reinhardtii.

In an embodiment of the present invention, vector P[1030], comprisingthe nucleotide sequence encoding the aadA gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Chlamydomonas reinhardtii to reflectthe codon bias inherent in nuclear genes of Chlamydomonas reinhardtii inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Chlamydomonas reinhardtii RbcS2 promoterupstream of the protein-coding sequence and operably linked to theChlamydomonas reinhardtii RbcS2 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Chlamydomonasreinhardtii genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic site of an endogenous lipid biosynthesis pathway gene.One skilled in the art can identify such homology regions within thesequence of the Chlamydomonas reinhardtii genome (referenced in thepublication by Merchant et al., Science, Vol. 318:5848 (2007), pp.245-250). Stable transformation of Chlamydomonas reinhardtii with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the aadA gene product may be used as a marker to select forChlamydomonas reinhardtii transformed with the transformation vector on,but not limited to, TAP agar medium comprising spectinomycin. Growthmedia suitable for Chlamydomonas reinhardtii lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Yantao et al. and Dent et al. Evaluation of fatty acid profiles ofChlamydomonas reinhardtii lipids may be assessed through standard lipidextraction and analytical methods described herein.

Example 31 Engineering Yarrowia lipolytica

Expression of recombinant genes in accordance with the present inventionin Yarrowia lipolytica may be accomplished by modifying the methods andvectors taught by Fickers et al. as discussed herein. Briefly, Fickerset al., Journal of Microbiological Methods, Vol. 55 (2003), pp. 727-737,reported the stable nuclear transformation of Yarrowia lipolytica withplasmid DNA. Using a lithium acetate transformation method, Fickersintroduced the plasmid JMP123 into Yarrowia lipolytica. Plasmid JMP123comprised a hygromycin B resistance cassette, comprising sequenceencoding the hygromycin B phosphotransferase gene product (hph),operably-linked to the Yarrowia lipolytica LIP2 gene promoter (GenBankAccession No. AJ012632) upstream of the hph protein-coding region andoperably linked to the Yarrowia lipolytica LIP2 gene 3′UTR/terminatordownstream of the hph protein-coding region. Prior to transformationwith JMP123, Yarrowia lipolytica were unable to propagate on mediumcomprising 100 ug/ml hygromycin. Upon transformation with JMP123,transformed Yarrowia lipolytica were obtained that were able topropagate on medium comprising 100 ug/ml hygromycin, therebyestablishing the hygromycin B antibiotic resistance cassette as aselectable marker for use in Yarrowia lipolytica. The nucleotidesequence provided on JMP123 of the promoter and 3 ‘UTR/terminator of theYarrowia lipolytica LIP2 gene served as donor sequences for homologousrecombination of the hph coding sequence into the LIP2 locus. Evaluationof the genomic DNA of the stable transformants was performed bySouthern. Fickers reported that selection and maintenance of thetransformed Yarrowia lipolytica was performed on agar plates comprisingstandard YPD medium (Yeast Extract Peptone Dextrose) with 100 ug/mlhygromycin. Liquid culturing of transformed Yarrowia lipolytica wasperformed in YPD medium with hygromycin. Other media and techniques usedfor culturing Yarrowia lipolytica have been reported and numerous otherplasmids, promoters, 3′ UTRs, and selectable markers for use in Yarrowialipolytica have been reported (for example see Pignede et al., Appliedand Environmental Biology, Vol. 66:8 (2000), pp. 3283-3289, Chuang etal., New Biotechnology, Vol. 27:4 (2010), pp. 277-282, and Barth andGaillardin, (1996), In: K,W. (Ed.), Nonconventional Yeasts inBiotecnology. Sprinter-Verlag, Berlin-Heidelber, pp. 313-388). Fickersreported that the transformation vector JMP123 and the Yarrowialipolytica LIP2 gene promoter and 3′ UTR/terminator are suitable toenable heterologous gene expression in Yarrowia lipolytica. In addition,Fickers reported that the hygromycin resistance cassette encoded onJMP123 was suitable for use as a selectable marker in Yarrowialipolytica.

In an embodiment of the present invention, vector JMP 123, comprisingthe nucleotide sequence encoding the hph gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Yarrowia lipolytica to reflect thecodon bias inherent in nuclear genes of Yarrowia lipolytica inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the Yarrowia lipolytica LIP2 gene promoterupstream of the protein-coding sequence and operably linked to theYarrowia lipolytica LIP2 gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Yarrowia lipolyticagenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Yarrowialipolytica genome (referenced in the publication by Dujun et al.,Nature, Vol. 430 (2004), pp. 35-44). Stable transformation of Yarrowialipolytica with the transformation vector is achieved through well-knowntransformation techniques including lithium acetate transformation orother known methods. Activity of the hph gene product may be used as amarker to select for Yarrowia lipolytica transformed with thetransformation vector on, but not limited to, YPD medium comprisinghygromycin. Growth media suitable for Yarrowia lipolytica lipidproduction include, but are not limited to, YPD medium, and thoseculture media described by Chuang et al. Evaluation of fatty acidprofiles of Yarrowia lipolytica lipids may be assessed through standardlipid extraction and analytical methods described herein.

Example 32 Engineering Mortierella alpine

Expression of recombinant genes in accordance with the present inventionin Mortierella alpine may be accomplished by modifying the methods andvectors taught by Mackenzie et al. as discussed herein. Briefly,Mackenzie et al., Applied and Environmental Microbiology, Vol. 66(2000), pp. 4655-4661, reported the stable nuclear transformation ofMortierella alpina with plasmid DNA. Using a protoplast transformationmethod, MacKenzie introduced the plasmid pD4 into Mortierella alpina.Plasmid pD4 comprised a hygromycin B resistance cassette, comprisingsequence encoding the hygromycin B phosphotransferase gene product(hpt), operably-linked to the Mortierella alpina histone H4.1 genepromoter (GenBank Accession No. AJ249812) upstream of the hptprotein-coding region and operably linked to the Aspergillus nidulansN-(5′-phophoribosyl)anthranilate isomerase (trpC) gene 3′UTR/terminatordownstream of the hpt protein-coding region. Prior to transformationwith pD4, Mortierella alpina were unable to propagate on mediumcomprising 300 ug/ml hygromycin. Upon transformation with pD4,transformed Mortierella alpina were obtained that were propagated onmedium comprising 300 ug/ml hygromycin, thereby establishing thehygromycin B antibiotic resistance cassette as a selectable marker foruse in Mortierella alpina. Evaluation of the genomic DNA of the stabletransformants was performed by Southern. Mackenzie reported thatselection and maintenance of the transformed Mortierella alpina wasperformed on PDA (potato dextrose agar) medium comprising hygromycin.Liquid culturing of transformed Mortierella alpina by Mackenzie wasperformed in PDA medium or in S2GYE medium (comprising 5% glucose, 0.5%yeast extract, 0.18% NH₄SO₄, 0.02% MgSO₄-7H₂O, 0.0001% FeCl₃-6H₂O, 0.1%,trace elements, 10 mM K₂HPO₄-NaH₂PO₄), with hygromycin. Other media andtechniques used for culturing Mortierella alpina have been reported andother plasmids, promoters, 3′ UTRs, and selectable markers for use inMortierella alpina have been reported (for example see Ando et al.,Applied and Environmental Biology, Vol. 75:17 (2009) pp. 5529-35 and Luet al., Applied Biochemistry and Biotechnology, Vol. 164:7 (2001), pp.979-90). Mackenzie reported that the transformation vector pD4 and theMortierella alpina histone H4.1 promoter and A. nidulans trpC gene 3′UTR/terminator are suitable to enable heterologous gene expression inMortierella alpina. In addition, Mackenzie reported that the hygromycinresistance cassette encoded on pD4 was suitable for use as a selectablemarker in Mortierella alpina.

In an embodiment of the present invention, vector pD4, comprising thenucleotide sequence encoding the hpt gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Mortierella alpina to reflect thecodon bias inherent in nuclear genes of Mortierella alpina in accordancewith Tables 24A-D. For each lipid biosynthesis pathway protein of Table25, the codon-optimized gene sequence can individually be operablylinked to the Mortierella alpina histone H4.1 gene promoter upstream ofthe protein-coding sequence and operably linked to the A. nidulans trpC3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Mortierella alpina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Mortierella alpina genome(referenced in the publication by Wang et al., PLOS One, Vol. 6:12(2011)). Stable transformation of Mortierella alpina with thetransformation vector is achieved through well-known transformationtechniques including protoplast transformation or other known methods.Activity of the hpt gene product may be used as a marker to select forMortierella alpina transformed with the transformation vector on, butnot limited to, PDA medium comprising hygromycin. Growth media suitablefor Mortierella alpina lipid production include, but are not limited to,S2GYE medium, and those culture media described by Lu et al. and Ando etal. Evaluation of fatty acid profiles of Mortierella alpina lipids maybe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 33 Engineering Rhodococcus opacus PD630

Expression of recombinant genes in accordance with the present inventionin Rhodococcus opacus PD630 may be accomplished by modifying the methodsand vectors taught by Kalscheuer et al. as discussed herein. Briefly,Kalscheuer et al., Applied and Environmental Microbiology, Vol. 52(1999), pp. 508-515, reported the stable transformation of Rhodococcusopacus with plasmid DNA. Using the transformation method ofelectroporation, Kalscheuer introduced the plasmid pNC9501 intoRhodococcus opacus PD630. Plasmid pNC9501 comprised a thiostreptonresistance (thio^(r)) cassette, comprising the full nucleotide sequenceof the Streptomyces azureus 23S rRNA A1067 methyltransferase gene,including the gene's promoter and 3′ terminator sequence. Prior totransformation with pNC9501, Rhodococcus opacus was unable to propagateon medium comprising 1 mg/ml thiostrepton. Upon transformation ofRhodococcus opacus PD630 with pNC9501, transformants were obtained thatpropagated on culture medium comprising 1 mg/ml thiostrepton, therebyestablishing the use of the thiostrepton resistance cassette as aselectable marker in Rhodococcus opacus PD630. A second plasmiddescribed by Kalscheuer, pAK68, comprised the resistance thio^(r)cassette as well as the gene sequences of the Ralstonia eutrophabeta-ketothiolase (phaB), acetoacetyl-CoA reductase (phaA), andpoly3-hydroxyalkanoic acid synthase (phaC) genes forpolyhydroxyalkanoate biosynthesis, driven by the lacZ promoter. UponpAK68 transformation of a Rhodococcus opacus PD630 strain deficient inpolyhydroxyalkanoate biosynthesis, transformed Rhodococcus opacus PD630were obtained that produced higher amounts of polyhydroxyalkanoates thanthe untransformed strain. Detectable activity of the introducedRalstonia eutropha phaB, phaA, and phaC enzymes indicted that theregulatory elements encoded on the pAK68 plasmid were suitable forheterologous gene expression in Rhodococcus opacus PD630. Kalscheuerreported that selection and maintenance of the transformed Rhodococcusopacus PD630 was performed on standard Luria Broth (LB) medium, nutrientbroth (NB), or mineral salts medium (MSM) comprising thiostrepton. Othermedia and techniques used for culturing Rhodococcus opacus PD630 havebeen described (for example see Kurosawa et al., Journal ofBiotechnology, Vol. 147:3-4 (2010), pp. 212-218 and Alverez et al.,Applied Microbial and Biotechnology, Vol. 54:2 (2000), pp. 218-223).Kalscheuer reported that the transformation vectors pNC9501 and pAK68,the promoters of the Streptomyces azureus 23S rRNA A1067methyltransferase gene and lacZ gene are suitable to enable heterologousgene expression in Rhodococcus opacus PD630. In addition, Kalscheuerreported that the thio^(r) cassette encoded on pNC9501 and pAK68 wassuitable for use as a selectable marker in Rhodococcus opacus PD630.

In an embodiment of the present invention, vector pNC9501, comprisingthe nucleotide sequence encoding the thio^(r) gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 25, each protein-coding sequencecodon-optimized for expression in Rhodococcus opacus PD630 to reflectthe codon bias inherent in nuclear genes of Rhodococcus opacus inaccordance with Tables 24A-D. For each lipid biosynthesis pathwayprotein of Table 25, the codon-optimized gene sequence can individuallybe operably linked to the lacZ gene promoter upstream of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Rhodococcus opacus PD630 genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Rhodococcusopacus PD630 genome (referenced in the publication by Holder et al.,PLOS Genetics, Vol. 7:9 (2011). Transformation of Rhodococcus opacusPD630 with the transformation vector is achieved through well-knowntransformation techniques including electoporation or other knownmethods. Activity of the Streptomyces azureus 23S rRNA A1067methyltransferase gene product may be used as a marker to select forRhodococcus opacus PD630 transformed with the transformation vector on,but not limited to, LB medium comprising thiostrepton. Growth mediasuitable Rhodococcus opacus PD630 lipid production include, but are notlimited to those culture media discussed by Kurosawa et al. and Alvarezet al. Evaluation of fatty acid profiles of Rhodococcus opacus PD630lipids may be assessed through standard lipid extraction and analyticalmethods described herein.

Example 34 Engineering Microalgae for Fatty Acid Auxotrophy

Prototheca moriformis (UTEX 1435) engineered to express a Cupheawrightii thioesterase (CwTE2), was used as the host organism for furthergenetic modification to knockout both endogenous thioesterase alleles,FATA1-1 and FATA1-2. Here, a first transformation construct wasgenerated to integrate a neomycin expression cassette at the FATA1-1locus. This construct, pSZ2226, included 5′ (SEQ ID NO: 150) and 3′ (SEQID NO: 151) homologous recombination targeting sequences (flanking theconstruct) to the FATA1-1 locus of the nuclear genome and a neomycinresistance protein-coding sequence under the control of the C.reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and the Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126). This NeoR expressioncassette is listed as SEQ ID NO: 135 and served as a selectable marker.

Upon transformation of pSZ2226, individual transformants were selectedon agar plates comprising sucrose and G418. A single isolate, Strain H,was selected for further genetic modification. A second transformationconstruct, pSZ2236, was generated to integrate polynucleotides enablingexpression of a thiamine selectable marker into Strain H at the FATA1-2locus. pSZ2236 included 5′ (SEQ ID NO: 152) and 3′ (SEQ ID NO: 153)homologous recombination targeting sequences (flanking the construct) tothe FATA1-2 genomic region for integration into the P. moriformisnuclear genome and an A. thaliana THIC protein coding region under thecontrol of the C. protothecoides actin promoter/5′UTR (SEQ ID NO: 142)and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126). This AtTHICexpression cassette is listed as SEQ ID NO: 143 and served as aselectable marker. Upon transformation of Strain H with pSZ2236 togenerate Strain I, individual transformants, were selected on agarplates comprising free fatty acids. Strain I was able to propagate onagar plates and in medium lacking thiamine and supplemented with freefatty acids.

Example 35 Engineering Microorganisms for Increased Production ofStearic Acid

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed with the plasmid construct pSZ2281 accordingto biolistic transformation methods as described herein and inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ2281 includedpolynucleotides encoding RNA hairpins (SAD2hpC, SEQ ID NO: 154) todown-regulate the expression of stearoyl-ACP desaturase, 5′ (SEQ ID NO:121) and 3′ (SEQ ID NO: 122) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the nuclear genome, and a S. cerevisiae suc2 sucroseinvertase coding region (SEQ ID NO: 124), to express the proteinsequence given in SEQ ID NO: 123, under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 125) and Chlorella vulgaris nitratereductase 3′ UTR (SEQ ID NO: 126). This S. cerevisiae suc2 expressioncassette is listed as SEQ ID NO: 127 and served as a selectable marker.The polynucleotide sequence encoding the SAD2hpC RNA hairpin was underthe control of the C. protothecoides actin promoter/5′UTR (SEQ ID NO:142) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126).

Upon transformation of Strain J with construct pSZ2281 therebygenerating Strain K, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed herein and inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass and analyzed using standard fatty acid methyl estergas chromatography flame ionization detection methods as described inExample 3 (also see PCT/US2012/023696). The fatty acid profiles(expressed as Area % of total fatty acids) of P. moriformis UTEX StrainJ propagated on glucose as a sole carbon source and three representativeisolates of Strain K, propagated on sucrose as a sole carbon source, arepresented in Table 26.

TABLE 26 Fatty acid profiles of Prototheca moriformis cells engineeredto express a hairpin RNA construct targeting stearoyl ACP desaturasegene/gene products. Area % Strain Strain Strain Strain Fatty acid J K-1K-2 K-3 C10:0 0.01 0.00 0.02 0.02 C12:0 0.03 0.05 0.05 0.05 C14:0 1.220.89 0.87 0.77 C16:0 26.75 29.23 28.96 27.55 C18:0 3.06 37.39 36.7636.41 C18:1 59.62 23.90 24.76 26.92 C18:2 7.33 5.44 5.54 5.54

The data presented in Table 26 show a clear impact of the expression ofa SAD2 hairpin RNA construct on the C18:0 and C18:1 fatty acid profilesof the transformed organism. The fatty acid profiles of Strain Ktransformants comprising a SAD2 hairpin RNA construct demonstrated anincrease in the percentage of saturated C18:0 fatty acids with aconcomitant diminution of unsaturated C18:1 fatty acids. Fatty acidprofiles of the untransformed strain comprise about 3% C18:0. Fatty acidprofiles of the transformed strains comprise about 37% C18:0. These dataillustrate the successful expression and use of polynucleotides enablingexpression of a SAD RNA hairpin construct in Prototheca moriformis toalter the percentage of saturated fatty acids in the engineered hostmicrobes, and in particular in increasing the concentration of C 18:0fatty acids and decreasing C 18:1 fatty acids in microbial cells.

Example 36 Engineering Microorganisms for Increased Production of OleicAcid Through Knockdown of an Endogenous Acyl-ACP Thioesterase

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed independently with each of the constructspSZ2402-pSZ2407 according to biolistic transformation methods asdescribed herein and in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of theconstructs pSZ2402-pSZ2407 included different polynucleotides encoding ahairpin RNA targeted against Prototheca moriformis FATA1 mRNAtranscripts to down-regulate the expression of fatty acyl-ACPthioesterase, 5′ (SEQ ID NO: 121) and 3′ (SEQ ID NO: 122) homologousrecombination targeting sequences (flanking the construct) to the 6Sgenomic region for integration into the nuclear genome, and a S.cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 124) toexpress the protein sequence given in SEQ ID NO: 123 under the controlof C. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 125) andChlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126). This S.cerevisiae suc2 expression cassette is listed as SEQ ID NO: 127 andserved as a selectable marker. Sequence listing identities for thepolynucleotides corresponding to each hairpin are listed in Table 27.The polynucleotide sequence encoding each RNA hairpin was under thecontrol of the C. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 125)and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 126).

TABLE 27 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain J. Plasmid Hairpin 1. construct 2. designation 3. SEQID NO: 4. pSZ2402 5. PmFATA-hpB 6. SEQ ID NO: 160 7. pSZ2403 8.PmFATA-hpC 9. SEQ ID NO: 161 10. pSZ2404 11. PmFATA-hpD 12. SEQ ID NO:162 13. pSZ2405 14. PmFATA-hpE 15. SEQ ID NO: 163 16. pSZ2406 17.PmFATA-hpF 18. SEQ ID NO: 164 19. pSZ2407 20. PmFATA-hpG 21. SEQ ID NO:165

Upon independent transformation of Strain J with each of the constructslisted in Table 27, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using standard fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 3.The fatty acidprofiles (expressed as Area % of total fatty acids) of P. moriformisUTEX Strain J propagated on glucose as a sole carbon source andrepresentative isolates of each transformation of Strain J, propagatedon sucrose as a sole carbon source, are presented in Table 28.

TABLE 28 Fatty acid profiles of Prototheca moriformis cells engineeredto express hairpin RNA constructs targeting fatty acyl-ACP thioesterasegene/gene products. Area % Fatty Acid Construct C10:0 C12:0 C14:0 C16:0C18:0 C18:1 C18:2 Strain J untransformed 0 0.05 1.32 26.66 3.1 59.077.39 0.04 0.07 1.36 24.88 2.24 61.92 6.84 0 0.08 1.33 25.34 2.39 61.726.5 PmFATA-hpB 0 0.07 1.29 25.44 2.26 61.7 6.69 0 0.06 1.33 25.1 2.3761.56 6.87 0 0.08 1.18 22.03 1.71 63.8 8.63 0 0.07 1.21 24.5 2.23 62.327.19 PmFATA-hpC 0 0.08 1.29 24.93 2.24 62.02 7.01 0.05 0.06 1.29 25.452.26 61.81 6.76 0 0.02 0.68 15.8 1.88 72.64 6.96 0 0.03 0.78 17.56 1.771.8 6.03 PmFATA-hpD 0 0.03 0.92 19.04 2.03 68.82 7.05 0 0.04 1.27 23.142.25 65.27 6.07 0 0.03 0.79 18.55 2.13 69.66 6.77 0 0.04 1.11 21.01 1.7465.18 8.55 PmFATA-hpE 0 0.03 1.08 21.11 1.54 64.76 8.87 0 0.03 1.1721.93 1.71 63.89 8.77 0.03 0.04 0.34 8.6 1.69 78.08 8.87 0 0.03 0.4910.2 1.52 76.97 8.78 PmFATA-hpF 0 0.03 1 20.47 2.22 66.34 7.45 0 0.031.03 21.61 1.88 65.39 7.76 0 0.03 1.03 20.57 2.36 64.73 8.75 PmFATA-hpG0 0.03 1.2 24.39 2.47 61.9 7.49 0 0.04 1.29 24.14 2.29 61.41 8.22

The data presented in Table 28 show a clear impact of the expression ofFATA hairpin RNA constructs on the C18:0 and C18:1 fatty acid profilesof the transformed organism. The fatty acid profiles of Strain Jtransformants comprising a FATA hairpin RNA construct demonstrated anincrease in the percentage of C18:1 fatty acids with a concomitantdiminution of C16:0 and C18:0 fatty acids. Fatty acid profiles of theuntransformed Strain J are about 26.66% C16:0, 3% C18:0, and about 59%C18:1 fatty acids. In contrast, the fatty acid profiles of thetransformed strains comprise as low as 8.6% C16:0 and 1.54% C18:0 andgreater than 78% C18:1 fatty acids. These data indicate that FATALenzyme of Prototheca morifomis (UTEX 1435) displays a preferentialspecificity for hydrolysis of fatty acids of length C18.

These data illustrate the utility and successful use of polynucleotideFATA RNA hairpin constructs in Prototheca moriformis to alter the fattyacids profile of engineered microbes, and in particular in increasingthe concentration of C18:1 fatty acids and decreasing C18:0 and C16:0fatty acids in microbial cells.

Example 37 Altering the Levels of Fatty Acids of Engineered MicrobesThrough Multiple Allelic Disruption of a Fatty Acid Desaturase

This example describes the use of a transformation vector to disrupt theFADc loci of Prototheca moriformis with a transformation cassettecomprising a selectable marker and sequence encoding an exogenous SADenzyme to engineer microorganisms in which the fatty acid profile of thetransformed microorganism has been altered.

A classically mutagenized (for higher oil production) derivative ofProtheca moriformis (UTEX 1435), strain A, was transformed with thetransformation construct pSZ1499 (SEQ ID NO: 246) according to biolistictransformation methods detailed in Example 2. pSZ1499 comprisednucleotide sequence of the Olea europaea stearoyl-ACP desaturase gene,codon-optimized for expression in Protheca moriformis UTEX 1435. ThepSZ1499 expression construct contained 5′ (SEQ ID NO: 247) and 3′ (SEQID NO: 248) homologous recombination targeting sequences (flanking theconstruct) to the FADc genomic region for integration into the nucleargenome and a S. cerevisiae suc2 sucrose invertase coding region underthe control of C. reinhardtii β-tubulin promoter/5′UTR and Chlorellavulgaris nitrate reductase 3′ UTR. This S. cerevisiae suc2 expressioncassette is listed as SEQ ID NO: 159 and served as a selection marker.The Olea europaea stearoyl-ACP desaturase coding region was under thecontrol of the Prototheca moriformis Amt03 promoter/5′UTR (SEQ ID NO:89) and C. vulgaris nitrate reductase 3′UTR, and the native transitpeptide was replaced with the Chlorella protothecoides stearoyl-ACPdesaturase transit peptide (SEQ ID NO: 49). The entire O. europaea SADexpression cassette was termed pSZ1499 and may be written as FADc5′btub-Suc2-nr amt03-5106SAD-OeSAD-nr-FADc3′.

Primary transformants were selected on plates containing sucrose as asole carbon source. Individual transformants were clonally purified andgrown under standard lipid production conditions at pH 7.0, similar tothe conditions as disclosed in Example 1. Fatty acid profiles wereanalyzed using standard fatty acid methyl ester gas chromatography flameionization (FAME GC/FID) detection methods as described in Example 3.The resulting fatty acid profiles from a set of representative clonesarising from the transformations of the transformation vector are shownin Table 29. Fatty acid profiles of lipids obtained from theuntransformed strain C strain grown under lipid production conditionscomprising glucose as a sole carbon source (pH 5.0) are additionallypresented in Table 29.

TABLE 29 Fatty acid profiles of Prototheca moriformis (UTEX 1435)multiply engineered to knockout endogenous FADc alleles and to expressan O. europaea stearoyl-ACP desaturase. % % % % Strain TransformantC16:0 C18:0 C18:1 C18:2 strain A untransformed 28.50 3.72 57.70 7.04strain A untransformed 28.57 3.69 57.61 7.07 strain A Transformant 120.37 1.13 74.38 0.01 pSZ1499 Transformant 2 19.98 1.16 74.60 0.00Transformant 3 20.10 1.16 74.70 0.00 Transformant 4 21.13 1.21 73.860.00 Transformant 5 19.95 1.11 74.58 0.00 Transformant 6 20.20 1.1474.61 0.00 Transformant 7 20.72 1.15 74.15 0.00 Transformant 8 20.061.11 74.44 0.00 Transformant 9 19.86 1.18 74.88 0.00

As shown in Table 29, transformation of strain C with pSZ1499 impactsthe fatty acid profiles of the transformed microbes. The untransformedPrototheca moriformis (UTEX 1435) strain C strain exhibits a fatty acidprofile comprising less than 60% C18:1 fatty acids and greater than 7%C18:2 fatty acids. In contrast, strain C strains transformed withpSZ1499 exhibited fatty acid profiles with an increased composition ofC18:1 fatty acids and a concomitant decrease in C18:0 and C18:2 fattyacids. C18:2 fatty acids were undetected in the fatty acid profiles ofstrain C transformed with pSZ1499. The absence of detectable C18:2 fattyacids in pSZ1499 transformants indicated that the transformation withpSZ1499, bearing homologous recombination targeting sequences forintegration into multiple FADc genomic loci, had abolished FAD activity.

Southern blot analysis was conducted to verify that multiple FADcalleles were interrupted by the pSZ1499 transformation vector. GenomicDNA was extracted from strain C and pSZ1499 transformants using standardmolecular biology methods. DNA from each sample was run on 0.8% agarosegels after digestion with the restriction enzyme PstI. DNA from this gelwas transferred onto a Nylon+ membrane (Amersham), which was thenhybridized with a P32-labeled polynucleotide probe corresponding to FADc3′ region. FIG. 1 shows maps of the pSZ1499 transformation cassette, thetwo sequenced FADc alleles of Prototheca moriformis (UTEX 1435), and thepredicted sizes of the alleles disrupted by the pSZ1499 transformationvector. FADc allele 1 comprises a PstI restriction site, whereas FADcallele 2 does not. Integration of the SAD cassette would introduce aPstI restriction site into the disrupted FADc allele, resulting in a ˜6kb fragment resolved on the Southern, regardless of which allele wasdisrupted. FIG. 2 shows the results of Southern blot analysis. Ahybridization band at ˜6 kb is detected in both transformants. Nosmaller hybridization bands, that would be indicative of uninterruptedalleles, were detected. These results indicate that both FADc alleleswere disrupted by pSZ1499.

The ablation of both alleles of the FADc fatty acid desaturase with aSAD expression cassette results in fatty acid profiles comprising about74% C18:1. Collectively, these data demonstrate the utility andeffectiveness of polynucleotides permitting knockout of FAD alleles andconcomitant exogenous expression of stearoyl-ACP desaturase enzymes toalter the fatty acid profile of engineered microorganisms.

All references cited herein, including patents, patent applications, andpublications, including Genbank Accession numbers, are herebyincorporated by reference in their entireties, whether previouslyspecifically incorporated or not. The publications mentioned herein arecited for the purpose of describing and disclosing reagents,methodologies and concepts that may be used in connection with thepresent invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein. In particular, the following patent applications arehereby incorporated by reference in their entireties for all purposes:PCT Application No. PCT/US2008/065563, filed Jun. 2, 2008, entitled“Production of Oil in Microorganisms”, PCT Application No.PCT/US2010/31108, filed Apr. 14, 2010, entitled “Methods of MicrobialOil Extraction and Separation”, PCT Publication No. WO 2010/063032,filed Nov. 30, 2009, entitled “Production of Tailored Oils inHeterotrophic Microorganisms”, PCT Application No. PCT/US2011/038463,filed May 27, 2011, entitled “Tailored Oils Produced From RecombinantHeterotrophic Microorganisms”, and PCT Application No.PCT/US/2012/023696, filed Feb. 2, 2012, entitled “Tailored Oils Producedfrom Recombinant Heterotrophic Microorganisms.”

SEQUENCE LISTING UTEX 329 Prototheca kruegani SEQ ID NO: 1TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGAAAATACTCTGGAGCCATAGCGAAAGCAAGTTTAGTAAGCTTAGGTCATTCTTTTTAGACCCGAAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTAGTAAAATAACATGGAGGCCCGAACCGACTAATGTTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAAAAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGCAGTAGCAGTACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGTTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGACCTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAGGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGGGTATGTCAAAACCTCCAGCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG UTEX 1440 Prototheca wickerhamii SEQ ID NO: 2TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGATTTAATAACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGTCAATTTAACAAAACTTTAAATAAATTATAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATGGTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGAAAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGGGGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCATAGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAAGTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTTAAAGAGTGCGTAATAGCTCACTGUTEX 1442 Prototheca stagnora SEQ ID NO: 3TGTTGAAGAATGAGCCGGCGAGTTAAAAAAAATGGCATGGTTAAAGATATTTCTCTGAAGCCATAGCGAAAGCAAGTTTTACAAGCTATAGTCATTTTTTTTAGACCCGAAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTCAAATAACATGGAGGCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAAAAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGCAGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGTTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGACCTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAGGCCCCAAAATGAAAATGATAGTGACTAAGGACGTGAGTATGTCAAAACCTCCAGCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG UTEX 288 Prototheca moriformis SEQ ID NO: 4TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGATAATTCTCTGGAGCCATAGCGAAAGCAAGTTTAACAAGCTAAAGTCACCCTTTTTAGACCCGAAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAGGCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAAAAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGCAGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTTCGAAAGTTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGACCTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAGGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGGGTATGTTAAAACCTCCAGCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTGUTEX 1439, UTEX 1441, UTEX 1435, UTEX 1437 Prototheca moriformisSEQ ID NO: 5TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGAATTAATAACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGCTAATTTAACAAAACATTAAATAAAATCTAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATGGTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGAAAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGGGGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCATAGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAAGTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTTAAAGAGTGCGTAATAGCTCACTGUTEX 1533 Prototheca wickerhamii SEQ ID NO: 6TGTTGAAGAATGAGCCGTCGACTTAAAATAAATGGCAGGCTAAGAGAATTAATAACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGCTAATTTAACAAAACATTAAATAAAATCTAAAGTCATTTATTTTAGACCCGAACCTGAGTGATCTAACCATGGTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAACCGACCGATGTTGAAAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGGGGTAAAGCACTGTTTCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACGATATATTAGTGAGACTATGGGGGATAAGCTCCATAGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAAGTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTTAAAGAGTGCGTAATAGCTCACTGUTEX 1434 Prototheca moriformis SEQ ID NO: 7TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCGTGGTTAAAGAAAATTCTCTGGAACCATAGCGAAAGCAAGTTTAACAAGCTTAAGTCACTTTTTTTAGACCCGAAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAGGCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAAAAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGCAGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTTGTGGGCTCCGAAAGTTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGACCTTGGGGGATAAGCTCCTTGGTCGAAAGGGAAACAGCCCAGATCACCAGTTAAGGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGAGTATGTCAAAACCTCCAGCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG UTEX 1438 Prototheca zopfii SEQ ID NO: 8TGTTGAAGAATGAGCCGGCGAGTTAAAAAGAGTGGCATGGTTAAAGAAAATTCTCTGGAGCCATAGCGAAAGCAAGTTTAACAAGCTTAAGTCACTTTTTTTAGACCCGAAACCGAGTGATCTACCCATGATCAGGGTGAAGTGTTGGTAAAATAACATGGAGGCCCGAACCGACTAATGGTGAAAAATTAGCGGATGAATTGTGGGTAGGGGCGAAAAACCAATCGAACTCGGAGTTAGCTGGTTCTCCCCGAAATGCGTTTAGGCGCAGCAGTAGCAACACAAATAGAGGGGTAAAGCACTGTTTCTTTCGTGGGCTTCGAAAGTTGTACCTCAAAGTGGCAAACTCTGAATACTCTATTTAGATATCTACTAGTGAGACCTTGGGGGATAAGCTCCTTGGTCAAAAGGGAAACAGCCCAGATCACCAGTTAAGGCCCCAAAATGAAAATGATAGTGACTAAGGATGTGAGTATGTCAAAACCTCCAGCAGGTTAGCTTAGAAGCAGCAATCCTTTCAAGAGTGCGTAATAGCTCACTG UTEX 1436 Prototheca moriformis SEQ ID NO: 9TGTTGAAGAATGAGCCGGCGACTTAGAAAAGGTGGCATGGTTAAGGAAATATTCCGAAGCCGTAGCAAAAGCGAGTCTGAATAGGGCGATAAAATATATTAATATTTAGAATCTAGTCATTTTTTCTAGACCCGAACCCGGGTGATCTAACCATGACCAGGATGAAGCTTGGGTGATACCAAGTGAAGGTCCGAACCGACCGATGTTGAAAAATCGGCGGATGAGTTGTGGTTAGCGGTGAAATACCAGTCGAACCCGGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAGTACATCTAGTCTATCTAGGGGTAAAGCACTGTTTCGGTGCGGGCTGTGAGAACGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACGATGTAGTAGTGAGACTGTGGGGGATAAGCTCCATTGTCAAGAGGGAAACAGCCCAGACCACCAGCTAAGGCCCCAAAATGGTAATGTAGTGACAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTTAAAGAGTGCGTAATAGCTCACTGHUP promoter from Chlorella (subsequence of GenBank accession numberX55349) SEQ ID NO: 10GATCAGACGGGCCTGACCTGCGAGATAATCAAGTGCTCGTAGGCAACCAACTCAGCAGCTGCTTGGTGTTGGGTCTGCAGGATAGTGTTGCAGGGCCCCAAGGACAGCAGGGGAACTTACACCTTGTCCCCGACCCAGTTTTATGGAGTGCATTGCCTCAAGAGCCTAGCCGGAGCGCTAGGCTACATACTTGCCGCACCGGTATGAGGGGATATAGTACTCGCACTGCGCTGTCTAGTGAGATGGGCAGTGCTGCCCATAAACAACTGGCTGCTCAGCCATTTGTTGGCGGACCATTCTGGGGGGGCCAGCAATGCCTGACTTTCGGGTAGGGTGAAAACTGAACAAAGACTACCAAAACAGAATTTCTTCCTCCTTGGAGGTAAGCGCAGGCCGGCCCGCCTGCGCCCACATGGCGCTCCGAACACCTCCATAGCTGTAAGGGCGCAAACATGGCCGGACTGTTGTCAGCACTCTTTCATGGCCATACAAGGTCATGTCGAGATTAGTGCTGAGTAAGACACTATCACCCCATGTTCGATTGAAGCCGTGACTTCATGCCAACCTGCCCCTGGGCGTAGCAGACGTATGCCATCATGACCACTAGCCGACATGCGCTGTCTTTTGCCACCAAAACAACTGGTACACCGCTCGAAGTCGTGCCGCACACCTCCGGGAGTGAGTCCGGCGACTCCTCCCCGGCGGGCCGCGGCCCTACCTGGGTAGGGTCGCCATACGCCCACGACCAAACGACGCAGGAGGGGATTGGGGTAGGGAATCCCAACCAGCCTAACCAAGACGGCACCTATAATAATAGGTGGGGGGACTAACAGCCCTATATCGCAAGCTTTGGGTGCCTATCTTGAGAAGCACGAGTTGGAGTGGCTGTGTACGGTCGACCCTAAGGTGGGTGTGCCGCAGCCTGAAACAAAGCGTCTAGCAGCTGCTTCTATAATGTGTCAGCCGTTGTGTTTCAGTTATATTGTATGCTATTGTTTGTTCGTGCTAGGGTGGCGCAGGCCCACCTACTGTGGCGGGCCATTGGTTGGTGCTTGAATTGCCTCACCATCTAAGGTCTGAACGCTCACTCAAACGCCTTTGTACAACTGCAGAACTTTCCTTGGCGCTGCAACTACAGTGTGCAAACCAGCACATAGCACTCCCTTACATCACCCAGCAGTACAACAChlorella ellipsoidea nitrate reductase promoter from AY307383SEQ ID NO: 11CGCTGCGCACCAGGGCCGCCAGCTCGCTGATGTCGCTCCAAATGCGGTCCCCCGATTTTTTGTTCTTCATCTTCTCCACCTTGGTGGCCTTCTTGGCCAGGGCCTTCAGCTGCATGCGCACAGACCGTTGAGCTCCTGATCAGCATCCTCAGGAGGCCCTTTGACAAGCAAGCCCCTGTGCAAGCCCATTCACGGGGTACCAGTGGTGCTGAGGTAGATGGGTTTGAAAAGGATTGCTCGGTCGATTGCTGCTCATGGAATTGGCATGTGCATGCATGTTCACAATATGCCACCAGGCTTTGGAGCAAGAGAGCATGAATGCCTTCAGGCAGGTTGAAAGTTCCTGGGGGTGAAGAGGCAGGGCCGAGGATTGGAGGAGGAAAGCATCAAGTCGTCGCTCATGCTCATGTTTTCAGTCAGAGTTTGCCAAGCTCACAGGAGCAGAGACAAGACTGGCTGCTCAGGTGTTGCATCGTGTGTGTGGTGGGGGGGGGGGGGTTAATACGGTACGAAATGCACTTGGAATTCCCACCTCATGCCAGCGGACCCACATGCTTGAATTCGAGGCCTGTGGGGTGAGAAATGCTCACTCTGCCCTCGTTGCTGAGGTACTTCAGGCCGCTGAGCTCAAAGTCGATGCCCTGCTCGTCTATCAGGGCCTGCACCTCTGGGCTGACCGGCTCAGCCTCCTTCGCGGGCATGGAGTAGGCGCCGGCAGCGTTCATGTCCGGGCCCAGGGCAGCGGTGGTGCCATAAATGTCGGTGATGGTGGGGAGGGGGGCCGTCGCCACACCATTGCCGTTGCTGGCTGACGCATGCACATGTGGCCTGGCTGGCACCGGCAGCACTGGTCTCCAGCCAGCCAGCAAGTGGCTGTTCAGGAAAGCGGCCATGTTGTTGGTCCCTGCGCATGTAATTCCCCAGATCAAAGGAGGGAACAGCTTGGATTTGATGTAGTGCCCAACCGGACTGAATGTGCGATGGCAGGTCCCTTTGAGTCTCCCGAATTACTAGCAGGGCACTGTGACCTAACGCAGCATGCCAACCGCAAAAAAATGATTGACAGAAAATGAAGCGGTGTGTCAATATTTGCTGTATTTATTCGTTTTAATCAGCAACCAAGTTCGAAACGCAACTATCGTGGTGATCAAGTGAACCTCATCAGACTTACCTCGTTCGGCAAGGAAACGGAGGCACCAAATTCCAATTTGATATTATCGCTTGCCAAGCTAGAGCTGATCTTTGGGAAACCAACTGCCAGACAGTGGACTGTGATGGAGTGCCCCGAGTGGTGGAGCCTCTTCGATTCGGTTAGTCATTACTAACGTGAACCCTCAGTGAAGGGACCATCAGACCAGAAAGACCAGATCTCCTCCTCGACACCGAGAGAGTGTTGCGGCAGTAGGACGACAAGYeast secretion signal SEQ ID NO: 12 MLLQAFLFLLAGFAAKISASHigher plants secretion signal SEQ ID NO: 13 MANKSLLLLLLLGSLASGConsensus eukaryotic secretion signal SEQ ID NO: 14 MARLPLAALGCombination higher plant/eukaryotic secretion signal SEQ ID NO: 15MANKLLLLLLLLLLPLAASG Codon-optimized yeast sucrose invertaseSEQ ID NO: 16ATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGA Yeast sucrose invertase, GenBank accession no. NP_012104.1SEQ ID NO: 17MTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK 5′donor DNA sequence of Prototheca moriformis delta 12 FAD knockouthomologous recombination targeting construct SEQ ID NO: 18GCTCTTCGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCGCCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCC 3′donor DNA sequence of Prototheca moriformis delta 12 FAD knockouthomologous recombination targeting construct SEQ ID NO: 19CAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGCATCGAGGTGGTCATCTCCGACCTCGCGTTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACATGATCGTGAACATGTGGCTGGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCCCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTACGCGGCGCCATGGCCACCGTCGACCGCTCCATGGGCCCGCCCTTCATGGACAGCATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCCGGCCCATCCTGGGCAAGTACTACCAATCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCCGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGCGCGCCTGCGCGAGGACGCAGAACAACGCTGCCGCCGTGTCTTTTGCACGCGCGACTCCGGCGCTTCGCTGGTGGCACCCCCATAAAGAAACCCTCAATTCTGTTTGTGGAAGACACGGTGTACCCCCACCCACCCACCTGCACCTCTATTATTGGTATTATTGACGCGGGAGTGGGCGTTGTACCCTACAACGTAGCTTCTCTAGTTTTCAGCTGGCTCCCACCATTGTAAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCPrototheca moriformis delta 12 FAD knockout homologous recombinationtargeting construct SEQ ID NO: 20GCTCTTCGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCGCCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGCATCGAGGTGGTCATCTCCGACCTCGCGTTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACATGATCGTGAACATGTGGCTGGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCCCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTACGCGGCGCCATGGCCACCGTCGACCGCTCCATGGGCCCGCCCTTCATGGACAGCATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCCGGCCCATCCTGGGCAAGTACTACCAATCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCCGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGCGCGCCTGCGCGAGGACGCAGAACAACGCTGCCGCCGTGTCTTTTGCACGCGCGACTCCGGCGCTTCGCTGGTGGCACCCCCATAAAGAAACCCTCAATTCTGTTTGTGGAAGACACGGTGTACCCCCACCCACCCACCTGCACCTCTATTATTGGTATTATTGACGCGGGAGTGGGCGTTGTACCCTACAACGTAGCTTCTCTAGTTTTCAGCTGGCTCCCACCATTGTAAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC 5′donor DNA sequence of Prototheca moriformis SAD2A knockout homologousrecombination targeting construct SEQ ID NO: 21GCTCTTCCGCCTGGAGCTGGTGCAGAGCATGGGTCAGTTTGCGGAGGAGAGGGTGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCCGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGACGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCCACCAAGTAGGTACC 3′donor DNA sequence of Prototheca moriformis SAD2A knockout homologousrecombination targeting construct SEQ ID NO: 22CAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAGGATCGTAGAGCTCCAGCCACGGCAACACCGCGCGCCTGGCGGCCGAGCACGGCGACAAGGGCCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCCGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTTCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCPrototheca moriformis SAD2A knockout homologous recombination targetingconstruct SEQ ID NO: 23GCTCTTCCGCCTGGAGCTGGTGCAGAGCATGGGTCAGTTTGCGGAGGAGAGGGTGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCCGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGACGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCCACCAAGTAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAGGATCGTAGAGCTCCAGCCACGGCAACACCGCGCGCCTGGCGGCCGAGCACGGCGACAAGGGCCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCCGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTTCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC 5′donor DNA sequence of Prototheca moriformis SAD2B knockout homologousrecombination targeting construct SEQ ID NO: 24GCTCTTCCCGCCTGGAGCTGGTGCAGAGCATGGGGCAGTTTGCGGAGGAGAGGGTGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCCGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGACGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCCACCAAGTAGGTACC 3′donor DNA sequence of Prototheca moriformis SAD2B knockout homologousrecombination targeting construct SEQ ID NO: 25CAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCACGGCGACAAGAACCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCPrototheca moriformis SAD2B knockout homologous recombination targetingconstruct SEQ ID NO: 26GCTCTTCCCGCCTGGAGCTGGTGCAGAGCATGGGGCAGTTTGCGGAGGAGAGGGTGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGCCCGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGACGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCCACCAAGTAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGACAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCACGGCGACAAGAACCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGAGAAGAGCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCPrimer 1  SEQ ID NO: 27 5′-TCACTTCATGCCGGCGGTCC-3′ Primer 2SEQ ID NO: 28 5′-GCGCTCCTGCTTGGCTCGAA-3′ S. cerevisiae suc2 cassetteSEQ ID NO: 29CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGA Chlamydomonas reinhardtii TUB2 promoter/5′ UTR SEQ ID NO: 30CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC Codon-optimized suc2 gene SEQ ID NO: 31ATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGA Chlorella vulgaris nitrate reductase 3′UTRSEQ ID NO: 32GCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCC pSZ1124 (FAD2B) 5′genomic targeting sequence SEQ ID NO: 33GCTCTTCGAGACGTGGTCTGAATCCTCCAGGCGGGTTTCCCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGAGCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCAATCCATAAACTCAAAACTGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCGCCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGGGTACCpSZ1124 (FAD2B) 3′ genomic targeting sequence SEQ ID NO: 34CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGTGAGTGAGAAGAGCpSZ1125 (FAD2C) 5′ genomic targeting sequence SEQ ID NO: 35GCTCTTCGAGGGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGACCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCACTCCATAAACTCAAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGGGTACCpSZ1125 (FAD2C) 3′ genomic targeting sequence SEQ ID NO: 36CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGTGAGTGAGAAGAGCamt03 promoter/UTR sequence SEQ ID NO: 37GGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCC 5′ 6S genomic donor sequence of Prototheca moriformisSEQ ID NO: 38GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACC3′6S genomic donor sequence of Prototheca moriformis SEQ ID NO: 39GAGCTCCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCFADc portion of the long hairpin RNA expression cassette from pSZ1468SEQ ID NO: 40ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATRelevant portion of the FADc long hairpin RNA expression cassette frompSZ1468 SEQ ID NO: 41GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATCTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCFADc portion of the long hairpin RNA expression cassette from pSZ1469SEQ ID NO: 42ACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATRelevant portion of the FADc long hairpin RNA expression cassette frompSZ1469 SEQ ID NO: 43GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACACTAGTATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATCTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCFADc portion of the long hairpin RNA expression cassette from pSZ1470SEQ ID NO: 44ACTAGTTCACTTGTGGAACCAGAGCGCGGAGTCGTCCTCGGGCGCGTCCGGGACGACGTAGCGGCAGTCGCGCCAGTCCTCCCACAGGGCGCGGCCGACCCAGCGGCTGTCGGACTGGTAGTACTTGCCCAGGATGGGCCTGATGGCGGCGGAGGCCTCCTCGGCGTGGTAGTGCGGGATGGTGCTGAAGAGGTGGTGCAGCACGTGGGTGTCGGAGATGTGGTGCAGGATGTTGTCCATGAAGGGCGGGCCCATGGAGCGGTCCACGGTGGCCATGGCGCCGCGCAGCCAGTCCCAGTCCTTCTCGAAGTAGTGCGGCAGCGCCGGGTGCGTGTGCTGGAGCAGCGTGATGAGCACGAGCCACATGTTCACGATCAGGTAGGGCACCACGTAGGTCTTGACCAGCCAGGCCCAGCCCATGGTGCGGCCCAGCACGCTGAGCCCGCTGAGCACCGCCACCAGCGCCAGGTCGGAGATGACCACCTCGATGCGCTCGCGCTTGCTGAAGATGGGCGACCACGGGTCAAAGTGGTTGGCGAAGCGCGGGTACGGCCGCGAGGCGACGTTGAACATGAGGTACAGCGGCCAGCCCAGGGTCAGGGTGACCAGCACCTTGCCCATGCGGATGGGCAGCCACTCCTCCCACTCCAGGCCCTCGTGCGCCACTGCGCGGTGCGGCGGCACAAACACCTCGTCCTTGTCCAGGCACCCCGTGTTGGAGTGGTGGCGGCGGTGCGAGTGCTTCCAGGAGTAGTAGGGCACCAGCAGCAGGCTGTGGAACACCAGGCCCACGCCGTCGTTGATGGCCTGGCTGGAGGAAAAGGCCTGGTGGCCGCACTCGTGCGCGCACACCCAGACACCCGTGCCGAAGGCGCCCTGGAAGGTGCCGGGAGGGTAGCCGGGGGGGTCAGCGGGACAGGCGCCTTCTCCTCCATGCGCGCCACCAGGACCTCAATACGGGCAACCAAAACCCTCAAACACACCTGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATRelevant portion of the FADc long hairpin RNA expression cassette frompSZ1470 SEQ ID NO: 45GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTTCACTTGTGGAACCAGAGCGCGGAGTCGTCCTCGGGCGCGTCCGGGACGACGTAGCGGCAGTCGCGCCAGTCCTCCCACAGGGCGCGGCCGACCCAGCGGCTGTCGGACTGGTAGTACTTGCCCAGGATGGGCCTGATGGCGGCGGAGGCCTCCTCGGCGTGGTAGTGCGGGATGGTGCTGAAGAGGTGGTGCAGCACGTGGGTGTCGGAGATGTGGTGCAGGATGTTGTCCATGAAGGGCGGGCCCATGGAGCGGTCCACGGTGGCCATGGCGCCGCGCAGCCAGTCCCAGTCCTTCTCGAAGTAGTGCGGCAGCGCCGGGTGCGTGTGCTGGAGCAGCGTGATGAGCACGAGCCACATGTTCACGATCAGGTAGGGCACCACGTAGGTCTTGACCAGCCAGGCCCAGCCCATGGTGCGGCCCAGCACGCTGAGCCCGCTGAGCACCGCCACCAGCGCCAGGTCGGAGATGACCACCTCGATGCGCTCGCGCTTGCTGAAGATGGGCGACCACGGGTCAAAGTGGTTGGCGAAGCGCGGGTACGGCCGCGAGGCGACGTTGAACATGAGGTACAGCGGCCAGCCCAGGGTCAGGGTGACCAGCACCTTGCCCATGCGGATGGGCAGCCACTCCTCCCACTCCAGGCCCTCGTGCGCCACTGCGCGGTGCGGCGGCACAAACACCTCGTCCTTGTCCAGGCACCCCGTGTTGGAGTGGTGGCGGCGGTGCGAGTGCTTCCAGGAGTAGTAGGGCACCAGCAGCAGGCTGTGGAACACCAGGCCCACGCCGTCGTTGATGGCCTGGCTGGAGGAAAAGGCCTGGTGGCCGCACTCGTGCGCGCACACCCAGACACCCGTGCCGAAGGCGCCCTGGAAGGTGCCGGGAGGGTAGCCGGGGGGGTCAGCGGGACAGGCGCCTTCTCCTCCATGCGCGCCACCAGGACCTCAATACGGGCAACCAAAACCCTCAAACACACCTGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATCTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCFADc portion of the long hairpin RNA expression cassette from pSZ1471SEQ ID NO: 46ACTAGTTCACTTGTGGAACCAGAGCGCGGAGTCGTCCTCGGGCGCGTCCGGGACGACGTAGCGGCAGTCGCGCCAGTCCTCCCACAGGGCGCGGCCGACCCAGCGGCTGTCGGACTGGTAGTACTTGCCCAGGATGGGCCTGATGGCGGCGGAGGCCTCCTCGGCGTGGTAGTGCGGGATGGTGCTGAAGAGGTGGTGCAGCACGTGGGTGTCGGAGATGTGGTGCAGGATGTTGTCCATGAAGGGCGGGCCCATGGAGCGGTCCACGGTGGCCATGGCGCCGCGCAGCCAGTCCCAGTCCTTCTCGAAGTAGTGCGGCAGCGCCGGGTGCGTGTGCTGGAGCAGCGTGATGAGCACGAGCCACATGTTCACGATCAGGTAGGGCACCACGTAGGTCTTGACCAGCCAGGCCCAGCCCATGGTGCGGCCCAGCACGCTGAGCCCGCTGAGCACCGCCACCAGCGCCAGGTCGGAGATGACCACCTCGATGCGCTCGCGCTTGCTGAAGATGGGCGACCACGGGTCAAAGTGGTTGGCGAAGCGCGGGTACGGCCGCGAGGCGACGTTGAACATGAGGTACAGCGGCCAGCCCAGGGTCAGGGTGACCAGCACCTTGCCCATGCGGATGGGCAGCCACTCCTCCCACTCCAGGCCCTCGTGCGCCACTGCGCGGTGCGGCGGCACAAACACCTCGTCCTTGTCCAGGCACCCCGTGTTGGAGTGGTGGCGGCGGTGCGAGTGCTTCCAGGAGTAGTAGGGCACCAGCAGCAGGCTGTGGAACACCAGGCCCACGCCGTCGTTGATGGCCTGGCTGGAGGAAAAGGCCTGGTGGCCGCACTCGTGCGCGCACACCCAGACACCCGTGCCGAAGGCGCCCTGGAAGGTGCCGGGAGGGTAGCCGGGGGGGTCAGCGGGACAGGCGCCTTCTCCTCCATGCGCGCCACCAGGACCTCAATACGGGCAACCAAAACCCTCAAACACACCTGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATRelevant portion of the FADc long hairpin RNA expression cassette frompSZ1471 SEQ ID NO: 47GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACACTAGTTCACTTGTGGAACCAGAGCGCGGAGTCGTCCTCGGGCGCGTCCGGGACGACGTAGCGGCAGTCGCGCCAGTCCTCCCACAGGGCGCGGCCGACCCAGCGGCTGTCGGACTGGTAGTACTTGCCCAGGATGGGCCTGATGGCGGCGGAGGCCTCCTCGGCGTGGTAGTGCGGGATGGTGCTGAAGAGGTGGTGCAGCACGTGGGTGTCGGAGATGTGGTGCAGGATGTTGTCCATGAAGGGCGGGCCCATGGAGCGGTCCACGGTGGCCATGGCGCCGCGCAGCCAGTCCCAGTCCTTCTCGAAGTAGTGCGGCAGCGCCGGGTGCGTGTGCTGGAGCAGCGTGATGAGCACGAGCCACATGTTCACGATCAGGTAGGGCACCACGTAGGTCTTGACCAGCCAGGCCCAGCCCATGGTGCGGCCCAGCACGCTGAGCCCGCTGAGCACCGCCACCAGCGCCAGGTCGGAGATGACCACCTCGATGCGCTCGCGCTTGCTGAAGATGGGCGACCACGGGTCAAAGTGGTTGGCGAAGCGCGGGTACGGCCGCGAGGCGACGTTGAACATGAGGTACAGCGGCCAGCCCAGGGTCAGGGTGACCAGCACCTTGCCCATGCGGATGGGCAGCCACTCCTCCCACTCCAGGCCCTCGTGCGCCACTGCGCGGTGCGGCGGCACAAACACCTCGTCCTTGTCCAGGCACCCCGTGTTGGAGTGGTGGCGGCGGTGCGAGTGCTTCCAGGAGTAGTAGGGCACCAGCAGCAGGCTGTGGAACACCAGGCCCACGCCGTCGTTGATGGCCTGGCTGGAGGAAAAGGCCTGGTGGCCGCACTCGTGCGCGCACACCCAGACACCCGTGCCGAAGGCGCCCTGGAAGGTGCCGGGAGGGTAGCCGGGGGGGTCAGCGGGACAGGCGCCTTCTCCTCCATGCGCGCCACCAGGACCTCAATACGGGCAACCAAAACCCTCAAACACACCTGGAAGAACCAGTAGAGCGGCCACATGATGCCGTACTTGACCCACGTAGGCACCGGTGCAGGGTCGATGTACGTCGACGCGACGTAGAGCAGGGACATGACCGCGATGTCAAAGGCCAGGTACATGCTGCTACGAAGCGCCGAGCGCTCGAAACAGTGCGCGGGGATGGCCTTGCGCAGCGTCCCGATCGTGAACGGAGGCTTCTCCACAGGCTGCCTGTTCGTCTTGATAGCCATCTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGC 5′donor DNA sequence of Prototheca moriformis FATA1 knockout homologousrecombination targeting construct SEQ ID NO: 48GCTCTTCGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGGGTACC 3′donor DNA sequence of Prototheca moriformis FATA1 knockout homologousrecombination targeting construct SEQ ID NO: 49GACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCTGGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCAATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCCCCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTATTCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCGGAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAATGGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTGAAGAGC SEQ ID NO: 50GCTCTTCGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCGTAGAGCTCACTAGTATCGATTTCGAAGACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCTGGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCAATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCCCCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTATTCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCGGAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAATGGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTGAAGAGC SEQ ID NO: 51GCTCTTCGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTATGGTGGTGGCCGCCGCCGCCAGCAGCGCCTTCTTCCCCGTGCCCGCCCCCCGCCCCACCCCCAAGCCCGGCAAGTTCGGCAACTGGCCCAGCAGCCTGAGCCAGCCCTTCAAGCCCAAGAGCAACCCCAACGGCCGCTTCCAGGTGAAGGCCAACGTGAGCCCCCACGGGCGCGCCCCCAAGGCCAACGGCAGCGCCGTGAGCCTGAAGTCCGGCAGCCTGAACACCCTGGAGGACCCCCCCAGCAGCCCCCCCCCCCGCACCTTCCTGAACCAGCTGCCCGACTGGAGCCGCCTGCGCACCGCCATCACCACCGTGTTCGTGGCCGCCGAGAAGCAGTTCACCCGCCTGGACCGCAAGAGCAAGCGCCCCGACATGCTGGTGGACTGGTTCGGCAGCGAGACCATCGTGCAGGACGGCCTGGTGTTCCGCGAGCGCTTCAGCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGACACCAGCCTGAACCACTGCAAGAGCGTGGGCCTGCTGAACGACGGCTTCGGCCGCACCCCCGAGATGTGCACCCGCGACCTGATCTGGGTGCTGACCAAGATGCAGATCGTGGTGAACCGCTACCCCACCTGGGGCGACACCGTGGAGATCAACAGCTGGTTCAGCCAGAGCGGCAAGATCGGCATGGGCCGCGAGTGGCTGATCAGCGACTGCAACACCGGCGAGATCCTGGTGCGCGCCACCAGCGCCTGGGCCATGATGAACCAGAAGACCCGCCGCTTCAGCAAGCTGCCCTGCGAGGTGCGCCAGGAGATCGCCCCCCACTTCGTGGACGCCCCCCCCGTGATCGAGGACAACGACCGCAAGCTGCACAAGTTCGACGTGAAGACCGGCGACAGCATCTGCAAGGGCCTGACCCCCGGCTGGAACGACTTCGACGTGAACCAGCACGTGAGCAACGTGAAGTACATCGGCTGGATTCTGGAGAGCATGCCCACCGAGGTGCTGGAGACCCAGGAGCTGTGCAGCCTGACCCTGGAGTACCGCCGCGAGTGCGGCCGCGAGAGCGTGGTGGAGAGCGTGACCAGCATGAACCCCAGCAAGGTGGGCGACCGCAGCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCGCCGACATCATGAAGGGCCGCACCGAGTGGCGCCCCAAGAACGCCGGCACCAACCGCGCCATCAGCACCTGATTAATTAACTCGAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAAGACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCTGGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCAATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCCCCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTATTCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCGGAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAATGGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTGAAGAGCCodon optimized sequence for Cuphea wrightii FatB2 (CwTE2) thioesteraseSEQ ID NO: 52ATGGTGGTGGCCGCCGCCGCCAGCAGCGCCTTCTTCCCCGTGCCCGCCCCCCGCCCCACCCCCAAGCCCGGCAAGTTCGGCAACTGGCCCAGCAGCCTGAGCCAGCCCTTCAAGCCCAAGAGCAACCCCAACGGCCGCTTCCAGGTGAAGGCCAACGTGAGCCCCCACGGGCGCGCCCCCAAGGCCAACGGCAGCGCCGTGAGCCTGAAGTCCGGCAGCCTGAACACCCTGGAGGACCCCCCCAGCAGCCCCCCCCCCCGCACCTTCCTGAACCAGCTGCCCGACTGGAGCCGCCTGCGCACCGCCATCACCACCGTGTTCGTGGCCGCCGAGAAGCAGTTCACCCGCCTGGACCGCAAGAGCAAGCGCCCCGACATGCTGGTGGACTGGTTCGGCAGCGAGACCATCGTGCAGGACGGCCTGGTGTTCCGCGAGCGCTTCAGCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGACACCAGCCTGAACCACTGCAAGAGCGTGGGCCTGCTGAACGACGGCTTCGGCCGCACCCCCGAGATGTGCACCCGCGACCTGATCTGGGTGCTGACCAAGATGCAGATCGTGGTGAACCGCTACCCCACCTGGGGCGACACCGTGGAGATCAACAGCTGGTTCAGCCAGAGCGGCAAGATCGGCATGGGCCGCGAGTGGCTGATCAGCGACTGCAACACCGGCGAGATCCTGGTGCGCGCCACCAGCGCCTGGGCCATGATGAACCAGAAGACCCGCCGCTTCAGCAAGCTGCCCTGCGAGGTGCGCCAGGAGATCGCCCCCCACTTCGTGGACGCCCCCCCCGTGATCGAGGACAACGACCGCAAGCTGCACAAGTTCGACGTGAAGACCGGCGACAGCATCTGCAAGGGCCTGACCCCCGGCTGGAACGACTTCGACGTGAACCAGCACGTGAGCAACGTGAAGTACATCGGCTGGATTCTGGAGAGCATGCCCACCGAGGTGCTGGAGACCCAGGAGCTGTGCAGCCTGACCCTGGAGTACCGCCGCGAGTGCGGCCGCGAGAGCGTGGTGGAGAGCGTGACCAGCATGAACCCCAGCAAGGTGGGCGACCGCAGCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCGCCGACATCATGAAGGGCCGCACCGAGTGGCGCCCCAAGAACGCCGGCACCAACCGCGCCATCAGCACCTGAProtein sequence for Cuphea wrightii FatB2 (CwTE2) thioesterase; GenBankAccession No. U56104 SEQ ID NO: 53MVVAAAASSAFFPVPAPRPTPKPGKFGNWPSSLSQPFKPKSNPNGRFQVKANVSPHPKANGSAVSLKSGSLNTLEDPPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLDRKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCKSVGLLNDGFGRTPEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGREWLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLHKFDVKTGDSICKGLTPGWNDFDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRESVVESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNRAISTCodon optimized Chlorella protothecoides (UTEX 250) stearoyl ACP desaturasetransit peptide cDNA sequence. SEQ ID NO: 54ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCCodon-optimized Glycine max KASII SEQ ID. NO: 55ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCTCCACCCACTCCGGCAAGACCATGGCCGTGGCCCTGCAGCCCACCCAGGAGATCACCACCATCAAGAAGCCCCCCACCAAGCAGCGCCGCGTGGTGGTGACCGGCCTGGGCGTGGTGACCCCCCTGGGCCACGAGCCCGACATCTTCTACAACAACCTGCTGGACGGCGCCTCCGGCATCTCCGAGATCGAGACCTTCGACTGCGCCGAGTACCCCACCCGCATCGCCGGCGAGATCAAGTCCTTCTCCACCGACGGCTGGGTGGCCCCCAAGCTGTCCAAGCGCATGGACAAGTTCATGCTGTACATGCTGACCGCCGGCAAGAAGGCCCTGGTGGACGGCGGCATCACCGACGACGTGATGGACGAGCTGAACAAGGAGAAGTGCGGCGTGCTGATCGGCTCCGCCATGGGCGGCATGAAGGTGTTCAACGACGCCATCGAGGCCCTGCGCATCTCCTACAAGAAGATGAACCCCTTCTGCGTGCCCTTCGCCACCACCAACATGGGCTCCGCCATGCTGGCCATGGACCTGGGCTGGATGGGCCCCAACTACTCCATCTCCACCGCCTGCGCCACCTCCAACTTCTGCATCCTGAACGCCGCCAACCACATCATCCGCGGCGAGGCCGACGTGATGCTGTGCGGCGGCTCCGACGCCGCCATCATCCCCATCGGCCTGGGCGGCTTCGTGGCCTGCCGCGCCCTGTCCCAGCGCAACACCGACCCCACCAAGGCCTCCCGCCCCTGGGACATCAACCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGTGCTGCTGCTGGAGGAGCTGGAGCACGCCAAGGAGCGCGGCGCCACCATCTACGCCGAGTTCCTGGGCGGCTCCTTCACCTGCGACGCCTACCACGTGACCGAGCCCCGCCCCGACGGCGCCGGCGTGATCCTGTGCATCGAGAAGGCCCTGGCCCAGTCCGGCGTGTCCAAGGAGGACGTGAACTACATCAACGCCCACGCCACCTCCACCCCCGCCGGCGACCTGAAGGAGTACCAGGCCCTGATGCACTGCTTCGGCCAGAACCCCGAGCTGCGCGTGAACTCCACCAAGTCCATGATCGGCCACCTGCTGGGCGCCGCCGGCGGCGTGGAGGCCGTGGCCACCATCCAGGCCATCCGCACCGGCTGGGTGCACCCCAACATCAACCTGGAGAACCCCGACAACGGCGTGGACGCCAAGGTGCTGGTGGGCTCCAAGAAGGAGCGCCTGGACGTGAAGGCCGCCCTGTCCAACTCCTTCGGCTTCGGCGGCCACAACTCCTCCATCATCTTCGCCCCCTACATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGA Codon-optimized Helianthus annuus KASII SEQ ID. NO: 56ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCTGCCGCCGCGGCGGCCGCGTGGCCATGGCCATCGCCATCCAGCCCTCCTCCATCGAGATGGAGGAGGAGACCACCCTGACCAAGCGCAAGCAGCCCCCCACCAAGCAGCGCCGCGTGGTGGTGACCGGCATGGGCGTGGAGACCCCCATCGGCAACAACCCCGACCAGTTCTACAACAACCTGCTGCAGGGCGTGTCCGGCATCACCCAGATCGAGGCCTTCGACTGCTCCTCCTACCCCACCCGCATCGCCGGCGAGATCAAGAACTTCTCCACCGACGGCTGGGTGGCCCCCAAGCTGTCCAAGCGCATGGACCGCTTCATGCTGTACATGCTGACCGCCGGCAAGAAGGCCCTGGCCGACGCCGGCATCTCCCCCTCCGACTCCGACGAGATCGACAAGTCCCGCTGCGGCGTGCTGATCGGCTCCGCCATGGGCGGCATGAAGGTGTTCAACGACGCCATCGAGGCCCTGCGCGTGTCCTACCGCAAGATGAACCCCTTCTGCGTGCCCTTCGCCACCACCAACATGGGCTCCGCCATGCTGGCCATGGACCTGGGCTGGATGGGCCCCAACTACTCCATCTCCACCGCCTGCGCCACCTCCAACTTCTGCATCCTGAACGCCGCCAACCACATCATCCGCGGCGAGGCCGACATGATGCTGTGCGGCGGCTCCGACGCCGTGATCATCCCCATCGGCCTGGGCGGCTTCGTGGCCTGCCGCGCCCTGTCCGAGCGCAACACCGACCCCGCCAAGGCCTCCCGCCCCTGGGACTCCGGCCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGTGCTGCTGCTGGAGGAGCTGGAGCACGCCAAGAAGCGCGGCGCCAAGATCTACGCCGAGTTCCTGGGCGGCTCCTTCACCTGCGACGCCTACCACATGACCGAGCCCCACCCCGAGGGCGCCGGCGTGATCCTGTGCATCGAGAAGGCCCTGTCCCAGGCCGGCGTGCGCCGCGAGGACGTGAACTACATCAACGCCCACGCCACCTCCACCCCCGCCGGCGACCTGAAGGAGTACCACGCCCTGCTGCACTGCTTCGGCAACAACCAGGAGCTGCGCGTGAACTCCACCAAGTCCATGATCGGCCACCTGCTGGGCGCCGCCGGCGCCGTGGAGGCCGTGGCCACCGTGCAGGCCATCCGCACCGGCTGGATCCACCCCAACATCAACCTGGAGAACCCCGACCAGGGCGTGGACACCAAGGTGCTGGTGGGCTCCAAGAAGGAGCGCCTGAACGTGAAGGTGGGCCTGTCCAACTCCTTCGGCTTCGGCGGCCACAACTCCTCCATCCTGTTCGCCCCCTTCCAGATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGA Codon-optimized Ricinus communis KASIISEQ ID. NO: 57ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCTCCCACTACTACTCCTCCAACGGCCTGTTCCCCAACACCCCCCTGCTGCCCAAGCGCCACCCCCGCCTGCACCACCGCCTGCCCCGCTCCGGCGAGGCCATGGCCGTGGCCGTGCAGCCCGAGAAGGAGGTGGCCACCAACAAGAAGCCCCTGATGAAGCAGCGCCGCGTGGTGGTGACCGGCATGGGCGTGGTGTCCCCCCTGGGCCACGACATCGACGTGTACTACAACAACCTGCTGGACGGCTCCTCCGGCATCTCCCAGATCGACTCCTTCGACTGCGCCCAGTTCCCCACCCGCATCGCCGGCGAGATCAAGTCCTTCTCCACCGACGGCTGGGTGGCCCCCAAGCTGTCCAAGCGCATGGACAAGTTCATGCTGTACATGCTGACCGCCGGCAAGAAGGCCCTGGCCGACGGCGGCATCACCGAGGACATGATGGACGAGCTGGACAAGGCCCGCTGCGGCGTGCTGATCGGCTCCGCCATGGGCGGCATGAAGGTGTTCAACGACGCCATCGAGGCCCTGCGCATCTCCTACCGCAAGATGAACCCCTTCTGCGTGCCCTTCGCCACCACCAACATGGGCTCCGCCATGCTGGCCATGGACCTGGGCTGGATGGGCCCCAACTACTCCATCTCCACCGCCTGCGCCACCTCCAACTTCTGCATCCTGAACGCCGCCAACCACATCATCCGCGGCGAGGCCGACATCATGCTGTGCGGCGGCTCCGACGCCGCCATCATCCCCATCGGCCTGGGCGGCTTCGTGGCCTGCCGCGCCCTGTCCCAGCGCAACGACGACCCCACCAAGGCCTCCCGCCCCTGGGACATGAACCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGTGCTGCTGCTGGAGGAGCTGGAGCACGCCAAGAAGCGCGGCGCCAACATCTACGCCGAGTTCCTGGGCGGCTCCTTCACCTGCGACGCCTACCACATGACCGAGCCCCGCCCCGACGGCGTGGGCGTGATCCTGTGCATCGAGAAGGCCCTGGCCCGCTCCGGCGTGTCCAAGGAGGAGGTGAACTACATCAACGCCCACGCCACCTCCACCCCCGCCGGCGACCTGAAGGAGTACGAGGCCCTGATGCGCTGCTTCTCCCAGAACCCCGACCTGCGCGTGAACTCCACCAAGTCCATGATCGGCCACCTGCTGGGCGCCGCCGGCGCCGTGGAGGCCATCGCCACCATCCAGGCCATCCGCACCGGCTGGGTGCACCCCAACATCAACCTGGAGAACCCCGAGGAGGGCGTGGACACCAAGGTGCTGGTGGGCCCCAAGAAGGAGCGCCTGGACATCAAGGTGGCCCTGTCCAACTCCTTCGGCTTCGGCGGCCACAACTCCTCCATCATCTTCGCCCCCTACAAGATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGARelevant portions of pSZ2041 including a codon-optimized Protothecamorifomis KASII SEQ ID NO: 58GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATCAATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAACTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCGCCGCCGCCGCCGACGCCAACCCCGCCCGCCCCGAGCGCCGCGTGGTGATCACCGGCCAGGGCGTGGTGACCTCCCTGGGCCAGACCATCGAGCAGTTCTACTCCTCCCTGCTGGAGGGCGTGTCCGGCATCTCCCAGATCCAGAAGTTCGACACCACCGGCTACACCACCACCATCGCCGGCGAGATCAAGTCCCTGCAGCTGGACCCCTACGTGCCCAAGCGCTGGGCCAAGCGCGTGGACGACGTGATCAAGTACGTGTACATCGCCGGCAAGCAGGCCCTGGAGTCCGCCGGCCTGCCCATCGAGGCCGCCGGCCTGGCCGGCGCCGGCCTGGACCCCGCCCTGTGCGGCGTGCTGATCGGCACCGCCATGGCCGGCATGACCTCCTTCGCCGCCGGCGTGGAGGCCCTGACCCGCGGCGGCGTGCGCAAGATGAACCCCTTCTGCATCCCCTTCTCCATCTCCAACATGGGCGGCGCCATGCTGGCCATGGACATCGGCTTCATGGGCCCCAACTACTCCATCTCCACCGCCTGCGCCACCGGCAACTACTGCATCCTGGGCGCCGCCGACCACATCCGCCGCGGCGACGCCAACGTGATGCTGGCCGGCGGCGCCGACGCCGCCATCATCCCCTCCGGCATCGGCGGCTTCATCGCCTGCAAGGCCCTGTCCAAGCGCAACGACGAGCCCGAGCGCGCCTCCCGCCCCTGGGACGCCGACCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGTGCTGGTGCTGGAGGAGCTGGAGCACGCCAAGCGCCGCGGCGCCACCATCCTGGCCGAGCTGGTGGGCGGCGCCGCCACCTCCGACGCCCACCACATGACCGAGCCCGACCCCCAGGGCCGCGGCGTGCGCCTGTGCCTGGAGCGCGCCCTGGAGCGCGCCCGCCTGGCCCCCGAGCGCGTGGGCTACGTGAACGCCCACGGCACCTCCACCCCCGCCGGCGACGTGGCCGAGTACCGCGCCATCCGCGCCGTGATCCCCCAGGACTCCCTGCGCATCAACTCCACCAAGTCCATGATCGGCCACCTGCTGGGCGGCGCCGGCGCCGTGGAGGCCGTGGCCGCCATCCAGGCCCTGCGCACCGGCTGGCTGCACCCCAACCTGAACCTGGAGAACCCCGCCCCCGGCGTGGACCCCGTGGTGCTGGTGGGCCCCCGCAAGGAGCGCGCCGAGGACCTGGACGTGGTGCTGTCCAACTCCTTCGGCTTCGGCGGCCACAACTCCTGCGTGATCTTCCGCAAGTACGACGAGATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAATCGATAGATCTCTTAAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTAATTAAGAGCTCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCPrototheca moriformis (UTEX 1435) SAD2 allele 2 SEQ ID NO: 59ATGGCGTCTGCCGTCACCTTTGCGTGCGCCCCTCCCCGCGGCGCGGTCGCCGCGCCGGGTCGCCGCGCTGCCTCGCGTCCCCTGGTGGTGCACGCCGTCGCCAGCGAGGCCCCGCTGGGCGTGCCGCCCTCGGTGCAGCGCCCCTCCCCCGTGGTCTACTCCAAGCTGGACAAGCAACACCGCCTGACGCCCGAGCGCCTGGAGCTGGTGCAGAGCATGGGTCAGTTGCGGAGGAGAGGGTGCTCCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTCCTGGCCCGACCCCGAGTCGCCCGACTTCGAGGACCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACCTTGGACGGTGTGCGCGACGACACGGGCGCGGCTGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCGACCAAGTACAGCCACGGCAACACCGCGCGCCTGGCGGCCGAGCACGGCGACAAGGGCCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCCGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTTCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGATCTCCGGACGCGAGATTATGGTCTAG Prototheca moriformis (UTEX 1435) SAD2 allele 2 SEQ ID NO: 60MASAVTFACAPPRGAVAAPGRRAASRPLVVHAVASEAPLGVPPSVQRPSPVVYSKLDKQHRLTPERLELVQSMGQLRRRGCSPCCTPWTSCGSRRTSWPDPESPDFEDQVAELRARAKDLPDEYFVVLVGDMITEEALPTYMAMLNTLDGVRDDTGAADHPWARWTRQWVAEENRHGDLLNKYCWLTGRVNMRAVEVTINNLIKSGMNPQTDNNPYLGFVYTSFQERATKYSHGNTARLAAEHGDKGLSKICGLIASDEGRHEIAYTRIVDEFFRLDPEGAVAAYANMMRKQITMPAHLMDDMGHGEANPGRNLFADFSAVAEKIDVYDAEDYCRILEHLNARWKVDERQVSGQAAADQEYVLGLPQRFRKLAEKTAAKRKRVARRPVAFSWISGREIMVPrototheca moriformis (UTEX 1435) SAD2 allele 1 SEQ ID NO: 61ATGGCGTCTGCCGTCACCTTTGCGTGCGCCCCTCCCCGCGGCGCGGTCGCCGCGCCGGGTCGCCGCGCTGCCTCGCGTCCCCTGGTGGTGCGCGCGGTCGCCAGCGAGGCCCCGCTGGGCGTTCCGCCCTCGGTGCAGCGCCCCTCCCCCGTGGTCTACTCCAAGCTGGACAAGCAGCACCGCCTGACGCCCGAGCGCCTGGAGCTGGTGCAGAGCATGGGGCAGTTTGCGGAGGAGAGGGTGCTGCCCGTGCTGCACCCCGTGGACAAGCTGTGGCAGCCGCAGGACTTTTTGCCCGACCCCGAGTCGCCCGACTTCGAGGATCAGGTGGCGGAGCTGCGCGCGCGCGCCAAGGACCTGCCCGACGAGTACTTTGTGGTGCTGGTGGGGGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGCTCAACACGCTGGACGGCGTGCGCGACGACACGGGCGCGGCCGACCACCCGTGGGCGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGCTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTATTTGGGGTTCGTCTACACCTCCTTCCAGGAGCGCGCCACCAAGTACAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCACGGCGACAAGAACCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCGCCGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCTGGATCTCCGGGCGCGAGATCATGGTCTAG Prototheca moriformis (UTEX 1435) SAD2 allele 1 SEQ ID NO: 62MASAVTFACAPPRGAVAAPGRRAASRPLVVRAVASEAPLGVPPSVQRPSPVVYSKLDKQHRLTPERLELVQSMGQFAEERVLPVLHPVDKLWQPQDFLPDPESPDFEDQVAELRARAKDLPDEYFVVLVGDMITEEALPTYMAMLNTLDGVRDDTGAADHPWARWTRQWVAEENRHGDLLNKYCWLTGRVNMRAVEVTINNLIKSGMNPQTDNNPYLGEVYTSFQERATKYSHGNTARLAAEHGDKNLSKICGLIASDEGRHEIAYTRIVDEFFRLDPEGAVAAYANMMRKQITMPAHLMDDMGHGEANPGRNLEADFSAVAEKIDVYDAEDYCRILEHLNARWKVDERQVSGQAAADQEYVLGLPQRFRKLAEKTAAKRKRVARRPVAFSWISGREIMVPrototheca moriformis (UTEX 1435) mitochondrial ACP allele 1SEQ ID NO: 63ATGGCGTTCTTGCAGCGGACGAGCGCGCTGGTGCGCCAGGGTGTCCTGTCTCGCCTCGCCGTGCAGGCCAGCCCCGCCCTGAACAGCGTCCGGGCCTTCGCCTCGGCGTCCTACCTGGACAAGAATGAGGTGACGCACCGCGTGCTGTCGATTGTCAAGAACTTTGACCGCGTCGACGCCGGCAAGGTCACGGACTCTGCCAACTTCCAGTCCGACCTGGGTCTGGACTCACTAGACACCGTGGAGCTGGTCATGGCCCTGGAGGAGGAGTTTGCGATCGAGATCCCGGATGCGGAGGCNGACAAGATCCTCTCCGTCCCGGAGGCGATTTCCTACATTGCCGCGAACCCCATGGCCAAGTAGPrototheca moriformis (UTEX 1435) Mitochondrial ACP allele 1SEQ ID NO: 64ATGGCGTTCTTGCAGCGGACGAGCGCGCTGGTGCGCCAGGGTGTCCTGTCTCGCCTCGCCGTGCAGGCCAGCCCCGCCCTGAACAGCGTCCGGGCCTTCGCCTCGGCGTCCTACCTGGACAAGAATGAGGTGACGCACCGCGTGCTGTCGATTGTCAAGAACTTTGACCGCGTCGACGCCGGCAAGGTCACGGACTCTGCCAACTTCCAGTCCGACCTGGGTCTGGACTCACTAGACACCGTGGAGCTGGTCATGGCCCTGGAGGAGGAGTTTGCGATCGAGATCCCGGATGCGGAGGCNGACAAGATCCTCTCCGTCCCGGAGGCGATTTCCTACATTGCCGCGAACCCCATGGCCAAGTAGPrototheca moriformis (UTEX 1435) Mitochondrial ACP allele 1SEQ ID NO: 65MAFLQRTSALVRQGVLSRLAVQASPALNSVRAFASASYLDKKEVTDRVLSIVKNFDRVDAGKVTDSANFQSDLGLDSLDTVELVMALEEEFAIEIPDAEADKILSVPEAISYIAANPMAKPrototheca moriformis (UTEX 1435) LPAAT-E SEQ ID NO: 66ATGGTTGCGGCTCAGGAAGACGGCTCCATGGAGAGGAGGACCTCGGCCAACCCCTCCGTGGGTCTTAGCAGCTTAAAGCACGTTCCGCTGAGTGCGGTGGACCTCCCGAATTTCGAGTCCGCTCCCAAGGGTGCGGCCAGCCCCGATGTCCATCGTCAGATAGAGGAACTGGTGGATAAGGCACGCGCAGATCGGTCACCGGCCTCGTTGTCGCTCCTAGCCGATGTGCTGGACATCAGCGGGCCGCTGCAGGACGCGGCCTCGGCGATGGTGGACGACTCCTTCCTGCGCTGCTTCACCTCCACCATGGACGAGCCCTGGAACTGGAACTTTTACCTGTTCCCGCTCTGGGCGCTCGGCGTCGTCGTCCGCAACCTGATCCTGTTCCCCCTGCGCCTCCTCACCATCGTGCTGGGGACGCTGCTCTTCGTGCTGGCCTTTGCCGTGACGGGCTTTCTCCCCAAGGAGAAGCGGCTCGCGGCCGAGCAGCGGTGCGTCCAGTTCATGGCGCAGGCGTTCGTGGCCTCCTGGACAGGCGTGATCCGGTACCACGGCCCCCGCCCCGTGGCGGCGCCCAACCGCGTGTGGGTCGCCAACCACACGTCCATGATCGACTACGCGGTGCTGTGCGCCTACTGCCCGTTTGCGGCCATCATGCAGCTGCACCCCGGCTGGGTGGGCGTCTTCCAGACGCGCTACCTGGCCTCGCTGGGCTGCCTCTGGTTCAACCGCACGCAGGCCAAGGACCGCACGCTCGTGGCCAAGCGCATGCGCGAGCACGTGGCCAGCGCGACCAGCACGCCGCTGCTCATCTTCCCCGAGGGCACCTGCGTCAACAACGAGTACTGCGTCATGTTCCGCAAGGGCGCCTTTGACCTGGGCGCCACCGTCTGCCCCGTGGCCATCAAGTACAACAAGATCTTCGTCGACGCCTTTTGGAACAGCAAGCGCCAGTCCTTCTCCGCGCACCTCATGAAGATCATGCGCTCGTGGGCCCTCGTCTGCGACGTCTACTTCCTGGAGCCCCAGACGCGGAGGGAGGGCGAGTCGGTCGAGGCGTTTGCGAACCGCPrototheca moriformis KASI allele 2, nucleotide sequence SEQ ID NO: 67ATGGTCGCGACCCTGTCCCTCGCCGGCCCCGCCTGCAACACGCAGTGCGTATCCAGCAAGCGGGTTGTCGCCTTCAACCGCCCCCATGTTGGCGTCCGGGCTCGATCAGCCGTGGTCGCCCGAGCTGCGAAGCGGGACCCCACCCAGCGCATTGTGATCACCGGAATGGGCGTGGCCTCCGTGTTTGGCAACGATGTCGAGACCTTTTACGACAAGCTTCTGGAAGGAACGAGCGGCGTGGACCTGATTTCCAGGTTTGACATCTCCGAGTTCCCGACCAAGTTTGCGGCGCAGATCACCGGCTTCTCCGTGGAGGACTGCGTGGACAAGAAGAACGCGCGGCGGTACGACGACGCGCTGTCGTACGCGATGGTGGCCTCCAAGAAGGCCCTGCGCCAGGCAGGCCTGGAGAAGGACAAGTGCCCCGAGGGCTACGGGGCGCTGGACAAGACGCGCACGGGCGTGCTGGTCGGCTCGGGCATGGGCGGGCTGACGGTCTTCCAGGACGGCGTCAAGGCGCTGGTGGAGAAGGGCTACAAGAAGATGAGCCCCTTCTTCATCCCCTACGCCATCACCAACATGGGCTCCGCGCTGGTGGGCATCGACCAGGGCTTCATGGGCCCCAACTACTCCGTCTCCACAGCCTGCGCGACGTCCAACTACGCATTTGTGAACGCGGCCAACCACATCCGCAAGGGCGACGCGGACGTCATGGTCGTCGGCGGCACCGAGGCCTCCATCGTGCCCGTGGGCCTGGGCGGCTTTGTGGCCTGCCGCGCGCTGTCCACGCGCAACGACGAGCCCAAGCGCGCGAGCCGGCCGTGGGACGAGGGCCGCGACGGCTTTGTGATGGGCGAGGGCGCGGCCGTGCTGGTCATGGAGTCGCTGGAGCACGCGCAGAAGCGTGGCGCGACCATCCTGGGCGAGTACCTGGGCGGCGCCATGACCTGCGACGCGCACCACATGACGGACCCGCACCCCGAGGGCCTGGGCGTGAGCACCTGCATCCGCCTGGCGCTCGAGGACGCCGGCGTCTCGCCCGACGAGGTCAACTACGTCAACGCGCACGCCACCTCCACCCTGGTGGGCGACAAGGCCGAGGTGCGCGCGGTCAAGTCGGTCTTTGGCGACATGAAGGGTATCAAGATGAACGCCACCAAGAGTATGATCGGGCACTGCCTGGGCGCCGCCGGCGGCATGGAGGCCGTCGCGACGCTCATGGCCATCCGCACCGGCTGGGTGCACCCCACCATCAACCACGACAACCCCATCGCCGAGGTCGATGGCCTGGACGTCGTCGCCAACGCCAAGGCCCAGCACGACATCAACGTCGCCATCTCCAACTCCTTCGGCTTTGGCGGGCACAACTCCGTCGTCGCCTTTGCGCCCTTCCGCGAGTAGPrototheca moriformis KASI allele 2, protein sequence SEQ ID NO: 68MVATLSLAGPACNTQCVSSKRVVAFNRPHVGVRARSAVVARAAKRDPTQRIVITGMGVASVEGNDVETFYDKLLEGTSGVDLISRFDISEFFTKFAAQITGFSVEDCVDKKNARRYDDALSYAMVASKKALRQAGLEKDKCPEGYGALDKTRTGVLVGSGMGGLTVFQDGVKALVEKGYKKMSPFFIPYAITNMGSALVGIDQGFMGPNYSVSTACATSNYAFVNAANHIRKGDADVMVVGGTEASIVPVGLGGEVACRALSTRNDEPKRASRPWDEGRDGFVMGEGAAVLVMESLEHAQKRGATILGEYLGGAMTCDAHHMTDPHPEGLGVSTCIRLALEDAGVSPDEVNYVNAHATSTLVGDKAEVRAVKSVFGDMKGIKMNATKSMIGHCLGAAGGMEAVATLMAIRTGWVHPTINHDNPIAEVDGLDVVANAKAQHDINVAISNSFGEGGHNSVVAFAPFRE*Prototheca moriformis (UTEX 1435) KASI allele 1, nucleotide sequenceSEQ ID NO: 69ATGGTCGCGACCCTCTCCCTCGCCGGCCCCGCCTGCAACACGCAGTGCGTATCCGGCAAGCGGGCTGTCGCCTTCAACCGCCCCCATGTTGGCGTCCGGGCTCGATCAGCCGTGGTCGCCCGGGCTGCGAAGCGGGACCCCACCCAGCGCATTGTGATCACCGGAATGGGCGTGGCCTCCGTGTTTGGCAACGATGTCGAGACCTTTTACAACAAGCTTCTGGAAGGAACGAGCGGCGTGGACCTGATTTCCAGGTTTGACATCTCCGAGTTCCCGACCAAGTTTGCGGCGCAGATCACCGGCTTCTCCGTGGAGGACTGCGTGGACAAGAAGAACGCGCGGCGGTACGACGACGCGCTGTCGTACGCGATGGTGGCCTCCAAGAAGGCCCTGCGCCAGGCGGGACTGGAGAAGGACAAGTGCCCCGAGGGCTACGGAGCGCTGGATAAGACGCGCGCGGGCGTGCTGGTCGGCTCGGGCATGGGCGGGCTGACGGTCTTCCAGGACGGCGTCAAGGCGCTGGTGGAGAAGGGCTACAAGAAGATGAGCCCCTTCTTCATCCCCTACGCCATCACCAACATGGGCTCCGCGCTGGTGGGCATCGACCAGGGCTTCATGGGGCCCAACTACTCCGTCTCCACGGCCTGCGCGACCTCCAACTACGCCTTTGTGAACGCGGCCAACCACATCCGCAAGGGCGACGCGGACGTCATGGTCGTGGGCGGCACCGAGGCCTCCATCGTGCCCGTGGGCCTGGGCGGCTTTGTGGCCTGCCGCGCGCTGTCCACGCGCAACGACGAGCCCAAGCGCGCGAGCCGGCCGTGGGACGAGGGCCGCGACGGCTTCGTGATGGGCGAGGGCGCGGCCGTGCTGGTCATGGAGTCGCTGGAGCACGCGCAGAAGCGCGGCGCGACCATCCTGGGCGAGTACCTGGGGGGCGCCATGACCTGCGACGCGCACCACATGACGGACCCGCACCCCGAGGGCCTGGGCGTGAGCACCTGCATCCGCCTGGCGCTCGAGGACGCCGGCGTCTCGCCCGACGAGGTCAACTACGTCAACGCGCACGCCACCTCCACCCTGGTGGGCGACAAGGCCGAGGTGCGCGCGGTCAAGTCGGTCTTTGGCGACATGAAGGGCATCAAGATGAACGCCACCAAGTCCATGATCGGGCACTGCCTGGGCGCCGCCGGCGGCATGGAGGCCGTCGCCACGCTCATGGCCATCCGCACCGGCTGGGTGCACCCCACCATCAACCACGACAACCCCATCGCCGAGGTCGACGGCCTGGACGTCGTCGCCAACGCCAAGGCCCAGCACAAAATCAACGTCGCCATCTCCAACTCCTTCGGCTTCGGCGGGCACAACTCCGTCGTCGCCTTTGCGCCCTTCCGCGAGTAGPrototheca moriformis (UTEX 1435) KASI allele 1, protein sequenceSEQ ID NO: 70MVATLSLAGPACNTQCVSGKRAVAFNRPHVGVRARSAVVARAAKRDPTQRIVITGMGVASVEGNDVETFYNKLLEGTSGVDLISRFDISEFFTKFAAQITGFSVEDCVDKKNARRYDDALSYAMVASKKALRQAGLEKDKCPEGYGALDKTRAGVLVGSGMGGLTVFQDGVKALVEKGYKKMSPFFIPYAITNMGSALVGIDQGFMGPNYSVSTACATSNYAFVNAANHIRKGDADVMVVGGTEASIVPVGLGGEVACRALSTRNDEPKRASRPWDEGRDGFVMGEGAAVLVMESLEHAQKRGATILGEYLGGAMTCDAHHMTDPHPEGLGVSTCIRLALEDAGVSPDEVNYVNAHATSTLVGDKAEVRAVKSVFGDMKGIKMNATKSMIGHCLGAAGGMEAVATLMAIRTGWVHPTINHDNPIAEVDGLDVVANAKAQHKINVAISNSFGFGGHNSVVAFAPFRE Prototheca moriformis (UTEX 1435) FATA allele 1SEQ ID NO: 71ATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGCATTCGGGGCAACGAGGTGGGCCCCTCGCAGCGGCTGACGATCACGGCGGTGGCCAACATCCTGCAGGAGGCGGCGGGCAACCACGCGGTGGCCATGTGGGGCCGGAGCTCGGAGGGTTTCGCGACGGACCCGGAGCTGCAGGAGGCGGGTCTCATCTTTGTGATGACGCGCATGCAGATCCAGATGTACCGCTACCCGCGCTGGGGCGACCTGATGCAGGTGGAGACCTGGTTCCAGACGGCGGGCAAGCTGGGCGCGCAGCGCGAGTGGGTGCTGCGCGACAAGCTGACCGGCGAGGCGCTGGGCGCGGCCACCTCGAGCTGGGTCATGATCAACATCCGCACGCGCCGGCCGTGCCGCATGCCGGAGCTCGTCCGCGTCAAGTCGGCCTTCTTCGCGCGCGAGCCGCCGCGCCTGGCGCTGCCGCCCGCGGTCACGCGTGCCAAGCTGCCCAACATCGCGACGCCGGCGCCGCTGCGCGGGCACCGCCAGGTCGCGCGCCGCACCGACATGGACATGAACGGGCACGTGAACAACGTGGCCTACCTGGCCTGGTGCCTGGAGGCCGTGCCCGAGCACGTCTTCAGCGACTACCACCTCTACCAGATGGAGATCGACTTCAAGGCCGAGTGCCACGCGGGCGACGTCATCTCCTCCCAGGCCGAGCAGATCCCGCCCCAGGAGGCGCTCACGCACAACGGCGCCGGCCGCAACCCCTCCTGCTTCGTCCATAGCATTCTGCGCGCCGAGACCGAGCTCGTCCGCGCGCGAACCACATGGTCGGCCCCCATCGACGCGCCCGCCGCCAAGCCGCCCAAGGCGAGCCACTGAPrototheca moriformis (UTEX 1435) FATA allele 1 SEQ ID NO: 72MAPTSLLASTGVSSASLWSSARSSACAFPVDHAVRGAPQRPLPMQRRCERTVAVRAAPAVAVRPEPAQEFWEQLEPCKMAEDKRIFLEEHRIRGNEVGPSQRLTITAVANILQEAAGNHAVAMWGRSSEGFATDPELQEAGLIFVMTRMQIQMYRYPRWGDLMQVETWFQTAGKLGAQREWVLRDKLTGEALGAATSSWVMINIRTRRPCRMPELVRVKSAFFAREPPRLALPPAVTRAKLPNIATPAPLRGHRQVARRTDMDMNGHVNNVAYLAWCLEAVPEHVESDYHLYQMEIDFKAECHAGDVISSQAEQIPPQEALTHNGAGRNPSCFVHSILRAETELVRARTTWSAPIDAPAAKPPKASHPrototheca moriformis (UTEX 1435) FAD linoleate Allele 2 SEQ ID NO: 73ATGGTCGCTGCGGGCGACGTCGCTGGAGCCACCGCCTCTGAGGGAGATGGCCGGGCCCTGGCCAACCCGCCGCCCTTCACGCTGGCCGACATCCGCAACGCCATTCCCAAGGACTGCTTCCGCAAGAGCGCGGCCAAGTCGTTGGCGTACCTGGGTCTGGACCTTTCCATCGTCACGGGCATGGCTGTGCTGGCCTACAAAATCAACTCGCCGTGGCTCTGGCCCCTGTACTGGTTTGCCCAGGGAACGATGTTTTGGGCCCTGTTTGTCGTCGGCCACGACTGTGGCCACCAGAGCTTTAGCACGAGCAAGCGGCTGAACGACGCGGTCGGTCTTTTCGTACACTCCATCATCGGCGTGCCGTACCACGGCTGGCGCATCTCCCACCGCACCCACCACAACAACCACGGCCACGTGGAGAACGACGAGTCCTGGTACCCGCCCACCGAGAGCGGGCTCAAGGCGATGACCGACATGGGCCGGCAGGGGCGCTTCCACTTTCCCACCATGCTCTTTGTCTACCCCTTTTACCTCTTCTGGCGGTCCCCGGGCAAGACCGGGTCCCACTTTAGCCCCTCCACCGACCTCTTCGCCTCGTGGGAGGCCCCCCTGATCCGGACGTCAAACGCGTGCCAGCTGGCGTGGCTGGGCGCGCTGGCGGCGGGGACCTGGGCGCTGGGCGTGCTGCCCATGCTCAACCTGTACCTCGCGCCCTACGTGATATCGGTGGCGTGGCTGGATCTGGTGACGTACCTGCACCACCACGGCCCCTCGGACCCGCGCGAGGAGATGCCCTGGTACCGCGGCCGCGAGTGGAGCTACATGCGCGGCGGGCTGACCACCATCGACCGCGACTACGGCCTCTTCAACAAGGTCCACCACGACATTGGGACGCACGTCGTGCACCACCTGTTCCCGCAGATCCCGCACTACAACCTCTGCCGCGCCACGGAGGCCGCCAAGAAGGTGCTGGGGCCCTACTACCGCGAGCCCGAGCGCTGCCCGCTGGGCCTGCTGCCCGTGCACCTGCTCGCGCCCCTGCTCCGCTCCCTCGGCCAGGACCACTTTGTCGACGACGCGGGCTCCGTCCTCTTCTACCGCCGCGCCGAGGGCATCAACCCCTGGATCCAGAAGCTCCTGCCCTPrototheca moriformis (UTEX 1435) FAD linoleate Allele 2 SEQ ID NO: 74MVAAGDVAGATASEGDGRALANPPPFTLADIRNAIPKDCFRKSAAKSLAYLGLDLSIVTGMAVLAYKINSPWLWPLYWFAQGTMFWALFVVGHDCGHQSFSTSKRLNDAVGLEVHSIIGVPYHGWRISHRTHHNNHGHVENDESWYPPTESGLKAMTDMGRQGRFHFPTMLFVYPFYLFWRSPGKTGSHFSPSTDLFASWEAPLIRTSNACQLAWLGALAAGTWALGVLPMLNLYLAPYVISVAWLDLVTYLHHHGPSDPREEMPWYRGREWSYMRGGLTTIDRDYGLENKVHHDIGTHVVHHLFPQIPHYNLCRATEAAKKVLGPYYREPERCPLGLLPVHLLAPLLRSLGQDHFVDDAGSVLFYRRAEGINPWIQKLLP Prototheca moriformis (UTEX 1435) FAD linoleate Allele 1SEQ ID NO: 75ATGGTCGCTGCGGGCGACGTCGCTAGAGCCACCGCCTCTGAGGGAGATGGCCGGGCCCTGGCCAACCCGCCGCCCTTCACGCTGGCCGACATCCGCAACGCCATTCCCAAGGACTGCTTCCGCAAGAGCGCGGCTAAGTCGTTGGCGTACCTGGGTTTGGACCTCTCCATCATCACGGGCATGGCTGTGCTGGCCTACAAAATCAACTCGCCGTGGCTCTGGCCCCTGTACTGGTTTGCCCAGGGAACGATGTTTTGGGCCCTGTTTGTCGTCGGACACGACTGTGGCCACCAGAGCTTTAGCACGAGCAAGCGGCTGAACGACGCGGTCGGTCTTTTTGTGCACTCCATCATCGGCGTGCCGTACCACGGCTGGCGCATCTCCCACCGCACCCACCACAACAACCACGGCCACGTGGAGAACGACGAGTCCTGCGAAAGCGGGCTCAAGGCGATGACCGATATGGGCCGGCAGGGCCGATTCCACTTTCCCTCCATGCTCTTTGTCTACCCCTTTTACCTCTTCTGGCGGTCCCCGGGCAAGACCGGGTCCCACTTTAGCCCCGCCACAGACCTCTTCGCCTTGTGGGAGGCCCCCCTGATCCGGACGTCCAACGCGTGCCAGCTGGCGTGGCTGGGCGCGCTGGCGGCGGGGACCTGGGCGCTGGGCGTGCTGCCGATGCTGAACCTGTACCTCGCGCCCTACGTGATCTCGGTGGCCTGGCTGGACCTGGTGACGTACCTGCACCACCACGGCCCCTCGGACCCGCGCGAGGAGATGCCCTGGTACCGCGGCCGGGAGTGGAGCTACATGCGCGGCGGGCTGACCACAATCGACCGCGACTACGGTCTTTTCAACAAGGTCCACCACGACATCGGGACGCACGTCGTGCACCACCTGTTCCCGCAGATCCCGCACTACAACCTCTGCCGCGCCACGAAGGCCGCCAAGAAGGTGCTGGGGCCCTACTACCGCGAGCCCGAGCGCTGCCCGCTGGGCCTGCTGCCCGTGCACCTGCTCGCGCCCCTGCTCCGCTCCCTCGGCCAGGACCACTTTGTCGACGACGCGGGCTCCGTCCTCTTCTACCGGCGCGCCGAGGGCATCAACCCCTGGATCCAGAAGCTCCTGCCCT Prototheca moriformis (UTEX 1435) FAD linoleate Allele 1SEQ ID NO: 76MVAAGDVARATASEGDGRALANPPPFTLADIRNAIPKDCFRKSAAKSLAYLGLDLSIITGMAVLAYKINSPWLWPLYWFAQGTMFWALFVVGHDCGHQSFSTSKRLNDAVGLFVHSIIGVPYHGWRISHRTHHNNHGHVENDESCESGLKAMTDMGRQGRFHFPSMLFVYPFYLFWRSPGKTGSHFSPATDLFALWEAPLIRTSNACQLAWLGALAAGTWALGVLPMLNLYLAPYVISVAWLDLVTYLHHHGPSDPREEMPWYRGREWSYMRGGLTTIDRDYGLFNKVHHDIGTHVVHHLFPQIPHYNLCRATKAAKKVLGPYYREPERCPLGLLPVHLLAPLLRSLGQDHFVDDAGSVLFYRRAEGINPWIQKLLPPrototheca moriformis (UTEX 1435) FAD-C delta-12 desaturase (FADc) Allele 2SEQ ID NO: 77MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGIMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVFHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVFVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMFNVASRPYPRFANHFDPWSPIFSKRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYMIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDSILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHKPrototheca moriformis (UTEX 1435) FADc allele 2 SEQ ID NO: 78ATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGTCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGTGCGGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCTTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCCCGCCCTTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTCGCGTTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACATGATCGTGAACATGTGGCTGGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCCCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTACGCGGCGCCATGGCCACCGTCGACCGCTCCATGGGCCCGCCCTTCATGGACAGCATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCCGGCCCATCCTGGGCAAGTACTACCAATCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCCGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAPrototheca moriformis (UTEX 1435) Oleate Desturase SEQ ID NO: 79ATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCGCCGGTGCCTACGTGGGTCAAGTATGGCGTCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTGTGAGGGTTGTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAPrototheca moriformis (UTEX 1435) FAD-B delta-12 desaturase (FADc Allele1), protein SEQ ID. NO: 80MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGVMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVFHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVFVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMFNVASRPYPRFANHFDPWSPIFSKRERIEVVISDLAXVAVLSGLSVLGRTMGWAWLVKTYVVPYLIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDNILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHKPrototheca moriformis (UTEX 1435) LPAAT-E SEQ ID. NO: 81ATGGTTGCGGCTCAGGAAGACGGCTCCATGGAGAGGAGGACCTCGGCCAACCCCTCCGTGGGTCTTAGCAGCTTAAAGCACGTTCCGCTGAGTGCGGTGGACCTCCCGAATTTCGAGTCCGCTCCCAAGGGTGCGGCCAGCCCCGATGTCCATCGTCAGATAGAGGAACTGGTGGATAAGGCACGCGCAGATCGGTCACCGGCCTCGTTGTCGCTCCTAGCCGATGTGCTGGACATCAGCGGGCCGCTGCAGGACGCGGCCTCGGCGATGGTGGACGACTCCTTCCTGCGCTGCTTCACCTCCACCATGGACGAGCCCTGGAACTGGAACTTTTACCTGTTCCCGCTCTGGGCGCTCGGCGTCGTCGTCCGCAACCTGATCCTGTTCCCCCTGCGCCTCCTCACCATCGTGCTGGGGACGCTGCTCTTCGTGCTGGCCTTTGCCGTGACGGGCTTTCTCCCCAAGGAGAAGCGGCTCGCGGCCGAGCAGCGGTGCGTCCAGTTCATGGCGCAGGCGTTCGTGGCCTCCTGGACAGGCGTGATCCGGTACCACGGCCCCCGCCCCGTGGCGGCGCCCAACCGCGTGTGGGTCGCCAACCACACGTCCATGATCGACTACGCGGTGCTGTGCGCCTACTGCCCGTTTGCGGCCATCATGCAGCTGCACCCCGGCTGGGTGGGCGTCTTCCAGACGCGCTACCTGGCCTCGCTGGGCTGCCTCTGGTTCAACCGCACGCAGGCCAAGGACCGCACGCTCGTGGCCAAGCGCATGCGCGAGCACGTGGCCAGCGCGACCAGCACGCCGCTGCTCATCTTCCCCGAGGGCACCTGCGTCAACAACGAGTACTGCGTCATGTTCCGCAAGGGCGCCTTTGACCTGGGCGCCACCGTCTGCCCCGTGGCCATCAAGTACAACAAGATCTTCGTCGACGCCTTTTGGAACAGCAAGCGCCAGTCCTTCTCCGCGCACCTCATGAAGATCATGCGCTCGTGGGCCCTCGTCTGCGACGTCTACTTCCTGGAGCCCCAGACGCGGAGGGAGGGCGAGTCGGTCGAGGCGTTTGCGAACCGC Prototheca moriformis (UTEX 1435) LPAAT-E, proteinSEQ ID. NO: 82MVAAQEDGSMERRTSANPSVGLSSLKHVPLSAVDLPNFESAPKGAASPDVHRQIEELVDKARADRSPASLSLLADVLDISGPLQDAASAMVDDSFLRCFTSTMDEPWNWNFYLFPLWALGVVVRNLILFPLRLLTIVLGTLLFVLAFAVTGFLPKEKRLAAEQRCVQFMAQAFVASWTGVIRYHGPRPVAAPNRVWVANHTSMIDYAVLCAYCPFAAIMQLHPGWVGVFQTRYLASLGCLWFNRTQAKDRTLVAKRMREHVASATSTPLLIFPEGTCVNNEYCVMFRKGAFDLGATVCPVAIKYNKIFVDAFWNSKRQSFSAHLMKIMRSWALVCDVYFLEPQTRREGESVEAFANRPrototheca moriformis (UTEX 1435) LPAAT-A, protein SEQ ID. NO: 83MPPPEAPTNGDLAAPFVRKDRFGEYGMAPQPPMVKVVLALEALVLLPLRAVSVFILVVLYWLICNASVALPPKYCAAVTVTAGRLVCRWALFCFGFHYIKWVNLAGAEEGPRPGGIVSNHCSYLDILLHMSDSFPAFVARQSTAKLPFIGIISQIMSCLYVNRDRSGPNHVGVADLVKQRMQDEAEGKTPPEYRPLLLFPEGTTSNGDYLLPFKTGAFLAGVPVQPVVLHYHRLPVYVPNEEEKADPKLYAQNVRKAMMEVAGTKDTTAVFEDKMRYLNSLKRKYGKPVPKKIEPrototheca moriformis (UTEX 1435) LPAAT-A SEQ ID. NO: 84AAATTATTGGCCTCATCCGCGAGGGTCGGAGCCTTTCAAACGTCACAAGCTGTATATTTGAGTCCCTCTCAATTCGTTTGAAATGAAGGAGCAAGTGGTTAATGCCCCCCCCTGAGGCCCCCACGAATGGGGACCTTGCGGCCCCCTTTGTGCGGAAGGACCGGTTCGGGGAGTATGGCATGGCCCCGCAGCCCCCGATGGTGAAGGTGGTGTTGGCACTCGAGGCCTTGGTGCTCTTGCCGCTGCGCGCGGTGAGCGTGTTCATCCTCGTCGTGCTATACTGGCTCATTTGCAATGCTTCGGTCGCACTCCCGCCCAAGTACTGCGCGGCCGTCACGGTCACCGCTGGGCGCTTGGTCTGCCGGTGGGCGCTCTTCTGCTTCGGATTCCACTACATCAAGTGGGTGAACCTGGCGGGCGCGGAGGAGGGCCCCCGCCCGGGCGGCATTGTTAGCAACCACTGCAGCTACCTGGACATCCTGCTGCACATGTCCGATTCCTTCCCCGCCTTTGTGGCGCGCCAGTCGACGGCCAAGCTGCCCTTTATCGGCATCATCAGCCAAATTATGAGCTGCCTCTACGTGAACCGCGACCGCTCGGGGCCCAACCACGTGGGTGTGGCCGACCTGGTGAAGCAGCGCATGCAGGACGAGGCCGAGGGGAAGACCCCGCCCGAGTACCGGCCGCTGCTCCTCTTCCCCGAGGGCACCACCTCCAACGGCGACTACCTGCTTCCCTTCAAGACCGGCGCCTTCCTGGCCGGGGTGCCCGTCCAGCCCGTCGTCCTCCACTACCACAGGTTGCCCGTGTACGTCCCCAATGAGGAGGAAAAGGCCGACCCCAAGCTGTACGCCCAAAACGTCCGCAAAGCCATGATGGAGGTCGCCGGGACCAAGGACACGACGGCGGTGTTTGAGGACAAGATGCGCTACCTGAACTCCCTGAAGAGAAAGTACGGCAAGCCTGTGCCTAAGAAAATTGAGTGAACCCCCGTCGTCGACCAAAGAGPrototheca moriformis (UTEX 1435) SAD1 allele 2, protein SEQ ID. NO: 85MASAAFTMSACPAMTGRAPEARRSGRPVATRLRAVAAPPRSQGLTPTIVRQDVLHSATDLQIEVIKDLTPVFESKILPLLPGVDDLWQPTDYLPASNSERFFDEIGELRERSASLSDELLVCLVGDMITEEALPTYMAMINTLDGMRDETGRDAHPYARWTRSWVAEENRHGDLLNKYLWLTGRVDMLAVERTIQRLISTGMDPGTENHPYHGFVFTSFQERATKLSHGATARIAHAAGDEALAKICGTIARDESRHEMAYTRTMEAIFERDPSGAVVAFAHMMLRKITMPAHLMDDGRHERATGRSLFDDYAAVAERIGVYTAKDYISILKHLIKRWKVEGLTGLTPEARQAQDLLCSLPTRFERLARHKSKKVTDTPPAQVQFSWVFQRPVAM Prototheca moriformis (UTEX 1435) SAD1 allele 2SEQ ID. NO: 86ATGGCTTCCGCGGCATTCACCATGTCGGCGTGCCCCGCGATGACTGGCAGGGCCCCTGAGGCACGTCGCTCCGGACGGCCAGTCGCCACCCGCCTGAGGGCTGTGGCGGCCCCGCCGCGTTCCCAGGGCCTCACCCCCACCATCGTCCGCCAAGATGTGTGCTGCACTCTGCCACGGACCTGCAGATCGAGGTGATCAAGGACCTGACCCCCGTGTTTGAGTCCAAGATCCTGCCCCTGCTGCCCGGCGTGGACGACCTGTGGCAGCCGACGGACTACCTTCCCGCCTCCAACTCGGAGCGTTTCTTCGACGAGATCGGGGAGCTGCGCGAGCGCTCGGCGTCGCTGTCGGACGAGCTTCTGGTGTGCCTGGTGGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGATCAACACGCTGGACGGGATGCGGGACGAGACGGGGCGCGACGCGCACCCGTACGCGCGCTGGACGCGCAGCTGGGTGGCGGAGGAGAACCGCCACGGCGACCTGCTGAACAAGTACCTCTGGCTGACGGGGCGCGTGGACATGCTGGCCGTGGAGCGCACGATCCAGCGCCTCATCTCCACGGGCATGGACCCGGGCACGGAGAACCACCCCTACCACGGCTTCGTCTTCACCTCCTTCCAGGAGCGCGCGACCAAGCTGAGCCACGGCGCCACCGCGCGCATCGCGCACGCCGCCGGCGACGAGGCGCTGGCCAAGATCTGCGGCACCATCGCGCGCGACGAGTCGCGCCACGAGATGGCCTACACGCGCACGATGGAGGCCATCTTCGAGCGCGACCCCTCGGGCGCGGTCGTCGCCTTTGCGCACATGATGCTGCGCAAGATCACCATGCCCGCGCACCTCATGGACGACGGCCGCCACGAGCGCGCCACGGGACGCTCGCTCTTCGACGACTACGCGGCCGTCGCCGAGCGCATCGGCGTCTACACCGCCAAGGACTACATCTCCATCCTCAAGCACCTCATCAAGCGCTGGAAGGTGGAGGGCCTGACCGGGCTGACCCCGGAGGCGCGCCAGGCGCAGGACCTGCTCTGCTCCCTCCCCACCCGCTTCGAGCGCCTGGCCAGGCACAAGTCCAAGAAGGTGACCGACACGCCCCCGGCCCAGGTCCAGTTCTCCTGGGTCTTCCAGAGGCCCGTCGCCATGTAAAGAGATTGGCGCCTGATTCGGTTTGGATCCGAGGATTTCCAATCGGTGAGAGGGACTGGGTGCCCAACTACCGCCCTTGCACCATCGCCCTTGCACTATTTATTCCCACTTTCTGCTCGCCCTGCCGGGCGATTGCGGGCGTTTCTGCCCTTGACGTATTAATTTCGCCCCTGCTGGCGCGAGGGTTCTTCAAGTTGATAAGAACGCACTCCCGCCAGCTCTGTACTTTTTCTGCGGGGCCCCTGCATGGCTTGTTCCCTATGCTTGCTCGATCGACGGCGCCCATTGCCCACGGCGTCGCCGCATCCATGTGAAGAAACACGG Prototheca moriformis (UTEX 1435) SAD1 allele 1SEQ ID. NO: 87ATGGCTTCCGCGGCATTCACCATGTCGGCGTGCCCCGCGATGACTGGCAGGGCCCCTGGGGCACGTCGCTCCGGACGGCCAGTCGCCACCCGCCTGAGGGCTGTGGCGGCCCCGCCGCGTTCCCAGGGCCTCACCCCCACCATCGTCCGCCAAGATGTGTGCTGCACTCTGCCACGGACCTGCAGATCGAGGTGATCAAGGACCTGACCCCCGTGTTTGAGTCCAAGATCCTGCCCCTGCTGCCCGGCGTGGACGACCTGTGGCAGCCGACGGACTACCTGCCCGCCTCCAACTCGGAGCGCTTCTTCGACGAGATCGGGGAGCTGCGCGAGCGCTCGGCGGCGCTGTCGGACGAGCTGCTGGTGTGCCTGGTCGGCGACATGATCACGGAGGAGGCGCTGCCGACCTACATGGCCATGATCAACACGCTGGACGGGATGCGGGACGAGACGGGGCGCGACGCGCACCCGTACGCGCGCTGGACACGCAGCTGGGTGGCGGAGGAGAACCGCCACGGCGACCTGCTGAACAAGTACCTCTGGCTGACGGGGCGCGTGGACATGCTGGCCGTGGAGCGCACCATCCAGCGCCTCATCTCCACGGGCATGGACCCGGGCACGGAGAACCACCCCTACCACGGCTTCGTCTTCACCTCCTTCCAGGAGCGCGCGACCAAGCTGAGCCACGGCGCCACCGCGCGCATCGCGCACGCCGCCGGCGACGAGGCCCTGGCCAAGATCTGCGGCACCATCGCGCGCGACGAGTCACGCCACGAGATGGCCTACACGCGCACGATGGAGGCCATCTTCGAGCGCGACCCCTCGGGCGCGGTCGTCGCCTTTGCGCACATGATGCTGCGCAAGATCACCATGCCCGCGCACCTCATGGACGACGGGCGGCACGAGCGCGCCACGGGGCGCTCGCTCTTCGACGACTACGCGGCCGTGGCCGAGCGCATCGGCGTCTACACCGCCAAGGACTACATCTCCATCCTCAAGCACCTCATCAAGCGCTGGAAGGTGGAGGGCCTGACCGGGCTGACCCCGGAGGCGCGCCAGGCCCAGGACCTGCTCTGCTCCCTCCCCGCCCGCTTTGAGCGCCTGGCCAAGCACAAGTCCAAGAAGGTGACCGACACGCCCCCGGCCCAGGTCCAGTTCTCCTGGGTCTTCCAGAGGCCCGTCGCCATGTAAAGTGGCAGAGATTGGCGCCTGATTCGATTTGGATCCAAGGATCTCCAATCGGTGATGGGGACTGAGTGCCCAACTACCACCCTTGCACTATCGTCCTCGCACTATTTATTCCCACCTTCTGCTCGCCCTGCCGGGCGATTGCGGGCGTTTCTGCCCTTGACGTATCAATTTCGCCCCTGCTGGCGCGAGGATTCTTCATTCTAATAAGAACTCACTCCCGCCAGCTCTGTACTTTTCCTGCGGGGCCCCTGCATGGCTTGTTCCCAATGCTTGCTCGATCGACGGCGCCCATTGCCCACGGCGCTGCCGCATCCATGTGAAGAAACACGG Prototheca moriformis (UTEX 1435) SAD1 allele 1SEQ ID. NO: 88MASAAFTMSACPAMTGRAPGARRSGRPVATRLRAVAAPPRSQGLTPTIVRQDVLHSATDLQIEVIKDLTPVFESKILPLLPGVDDLWQPTDYLPASNSERFFDEIGELRERSAALSDELLVCLVGDMITEEALPTYMAMINTLDGMRDETGRDAHPYARWTRSWVAEENRHGDLLNKYLWLTGRVDMLAVERTIQRLISTGMDPGTENHPYHGFVFTSFQERATKLSHGATARIAHAAGDEALAKICGTIARDESRHEMAYTRTMEAIFERDPSGAVVAFAHMMLRKITMPAHLMDDGRHERATGRSLFDDYAAVAERIGVYTAKDYISILKHLIKRWKVEGLTGLTPEARQAQDLLCSLPARFERLAKHKSKKVTDTPPAQVQFSWVFQRPVAMPrototheca moriformis (UTEX 1435) Plastidial ACP allele 1 SEQ ID. NO: 89MAMSMTSCRAVCAPRATLRVQAPRVAVRPFRAQRMICRAVDKASVLSDVRVIIAEQLGTDVEKVNADAKFADLGADSLDTVEIMMALEEKFDLQLDEEGAEKITTVQEAADLISAQIGAPrototheca moriformis (UTEX 1435) Plastidial ACP allele 2 SEQ ID. NO: 90MAMSMTSCRAVCAPRATLRVQAPRVAVRPFRAQRMICRAVDKASVLSDVRVIIAEQLGTDVEKVNADAKFADLGADSLDTVEXMMALEEKFDLQLDEEGAEKITTVQEAADLISAQXGAPrototheca moriformis (UTEX 1435) Acetyl-CoA carboxylase alpha-CT subunitallele 2 SEQ ID. NO: 91ATGCTGCTCTCGCGCTTCAACCCCATCAGCGAGAAGGCCACCAACGCCACGGTGCTCGACTTTGAGAAACCTTTGGTGGAACTTGATCATAAAATTCGCGAGGTCCGCAAAGTCGCGGAGGAGAACGGCGTGGACGTCACGGAGCAGATCCGCGAGCTGGAGGACCGCGCGCAGCAGGTAGTCGAGTCTGTGTGGAGAGGGTTGCGAAAGGAGACGTACTCCAAGCTGACGCCGATCCAGCGGCTGCAGGTGGCGCGCCACCCCAACCGCCCCACTTTCTTGGACATTGCGCTCAACATCACGGACAAGTTTGTGGAGCTGCACGGCGATCGCGCGGGGTACGACGACCCGGCGCTCGTCTGCGGCATCGCGTCGATCGACAACGTGAGCTTCATGTTCATGGGCCACCAGAAGGGCCGCAACACCAAGGAGAACATCTACCGCAATTTCGGCATGCCGCAGCCCAACGGCTACCGCAAGGCGCTGCGCTTCATGCGCCTGGCGGACAAGTTTGGCTTCCCCATCGTCACGTTTGTGGACACGCCCGGCGCGTACGCGGGGCTCAAGGCCGAGGAGCTGGGCCAGGGCGAGGCCATCGCGGTCAACCTGCGCGAGATGTTTGGCTTCCGCGTGCCCATCATCTCCATCGTCATCGGCGAGGGCGGCTCCGGCGGCGCGCTGGCCATCGGCTGCGCCAACCGCATGCTCATCATGGAGAACGCGGTGTACTACGTGGCCTCTCCCGAGGCCTGCGCGGCCATCCTGTGGAAGAGCCGCGACAAGGCGGGCCTGGCCACCGAGGCGCTCAAGATCACGCCCGCCGACCTGGTCAAGCTCAAGGTCATGGACGAGATCGTGCCGGAGCCGCTGGGCGCCGCGCACACCGACCCCATGGGCGCCTTCCCGGCCGTGCGCGAGGCCATCCTGCGCCACPrototheca moriformis Acetyl-CoA carboxylase alpha-CT subunit allele 1SEQ ID. NO: 92ATGTTCAATAGATACTATCTTGCAACAAGATTGCACTTGGGTTTGGTCGTCTGCTGTTGTGCACTACCATGTGACGTTGTGCTCGTCCATTCTGTTCCCGTCCAGGTCCGCAAAGTCGCGGAGGAGAACGGCGTGGACGTCACGGAGCAGATCCGCGAGCTGGAGGACCGCGCGCAGCAGGTAGTCGAGTCTGTGTGGAGAGGGGTGGGATACTTTGGAAGAATGGGTGCTGTGGAGTCCAGTCATTTGAAGGAAAGGGTAGTCGACTTCCCTCTGTGGTCGTATCTATCTTCTCCAAGCATATCTCACTTTGCCCCTCTTCTCCGCCCCCCTCCATTACCAACCCGTCCACAGTTGCGAAAGGAGACGTACTCCAAGCTGACGCCGATCCAGCGGCTGCAGGTGGCGCGCCACCCCAACCGCCCCACTTTCTTGGACATTGCGCTCAACATCACGGACAAGTTTGTGGAGCTGCACGGCGATCGCGCGGGGTACGACGACCCGNNNNNNNNNNAGAACGGCGTGGACGTCACGGAGCAGATCCGCGAGCTGGAGGACCGCGCGCAGCAGTGTAGTCAACTTTTCTGTGGTTCTATCCATCTTCCCCAAGTATATCTCACCCCCTCTTCCCCCCCTCTCCCCTTTAACAACACCCCGTCCACAGTTGCGAAAGGAGACATACTCCAAGCTGACGCCGATCCAGCGGCTGCAGGTGGCGCGCCACCCCAACCGCCCTACTTTCTTGGACATTGCGCTCAACATCACGGACAAGTTTGTGGAGCTGCACGGCGACCGCGCGGGGTACGACGACCCCGCGCTCGTCTGCGGCATCGCGTCGATCGACAACGTGAGCTTCATGTTCATGGGCCACCAGAAGGGCCGCAACACCAAGGAGAACATCTACCGCAATTTCGGCATGCCGCAGCCCAACGGCTACCGCAAGGCGCTGCGCTTCATGCGCCTGGCGNNNNNNNNNNCGACAACGTGAGCTTCATGTTCATGGGCCACCAGAAGGGCCGCAACACCAAGGAGAACATCTACCGAAACTTTGGCATGCCGCAGCCCAACGGCTACCGCAAGGCGCTGCGCTTCATGCGCCTGGCCGACAAGTTTGGCTTCCCCATCGTCACGTTTGTGGACACGCCCGGCGCGTACGCGGGGCTCAAGGCCGAGGAGCTGGGCCAGGGCGAGGCCATCGCGGTCAACCTGCGCGAGATGTTTGGCTTCCGCGTGCCCATCATCTCCATCGTCATCGGCGAGGGCGGCTCCGGCGGCGCGCTGGCCATCGGCTGCGCCAACCGCATGCTCATCATGGAGAACGCGGTGTACTACGTGGCCTCTCCCGAGGCCTGCGCGGCCATCCTGTGGAAGAGCCGCGACAAGGCGGGCCTGGCCACCGAGGCGCTCAAGATCACGCCCGCCGACCTGGTCAAGCTCAAGGTCATGGACGAGATCGTGCCGGAGCCGCTGGGCGCCGCGCACACCGACCCCATGGGCGCCTTCCCGGCCGAGGAGGCCGACTACATCGAGCAGATGGCCGAGCGCTGGCGGTGGTGGGACAGCCTGCTGGAGGACAAGAAGGACATCGTCAACCCGCCAAAGACCGAGCACCCGGGCTGGCTCATCCCGGGCTTTGCCGAGTTCGCCATCGGCGTCGCCAAGGCCGCCGACGCGCGCCAGGCGCGCATGGGGCTCGAGGGCGAGAGCGCCGCGCACGCCAACGACGCCGGCATCGGCACCGAGAGGGACGGCGGCGCCGAGGGCAACGGCGCGAACGCCGAGAACGGGGCGCACGACGAGTCGTCTATCGCGGAGACCGAGTGAPrototheca moriformis (UTEX 1435) 1b-ACCase-BC-A SEQ ID. NO: 93MAPASVNLTQVCGGANVEEPSVPQGDMGDHGLIQHVDSIAFRATPCPAAVAGRRQFPRGAGPSRRSQRSTGSARLTPSASTPSRLVARAETGTNGSAALTPIHKVLIANRGEIAVRVIRACKELGIKTVAVYSTADRECLHAQLADEAVCIGEAPSSESYLNIPTILSAAMSRGADAIHPGYGELSENAEFVDICKDHGINFIGPGSEHIRVMGDKATARETMKQAGVPTVPGSDGLVETPEEALEVADQVGFPVMIKATAGGGGRGMRLAMTRDEFLPLLRAAQGEAQAAFGNGAVYLERYVKDPRHIEFQVLADKHGNVIHLGERDCSIQRRNQKLLEEAPSPALTPEVRAAMGEAATNAARSIGYVGVGTIEFLWEPAGFYFMEMNTRIXXXXCARSGHSGVCPGGAGETQAWRAIAHSPGTKEVIGWVCKDSKERPFKRPPRLAGRALESSSNSSTGAASRSAANSVGDGINIQRGGARANRLSKENNTQETRAPFGQTKRERRLEGQVGTQAVEGAPRHMPrototheca moriformis Heteromeric acetyl-CoA carboxylase BC subunit allele2 SEQ ID. NO: 94MAPASVNLTQVCCSAGHGLRPARATPCPAAVAGRRQFPRGAGPSRRSQRSTGSARLTPSASTPSRLVARAETGTNGSAALTPIHKVLIANRGEIAVRVIRACKELGIKTVAVYSTADRECLHAQLADEAVCIGEAPSSESYLNIPTILSAAMSRGADAIHPGYGELSENAEFVDICKDHGINFIGPGSEHIRVMGDKATARETMKQAGVPTVPGSDGLVETPEEALEVADQVGFPVMIKATAGGGGRGMRLAMTRDEFLPLLRAAQGEAQAAFGNGAVYLERYVKDPRHIEFQVLADKHGNVIHLGERDCSIQRRNQKLLEEAPSPALTPEVRAAMGEAATNAARSIGYVGVGTIEFLWEPAGFYFMEMNTRIQVEHPVTEMITGVDLIQEQIRAAMGEPLRLTQEDIVIKGHSIECRINAEDPFKGFRPGPGRVTAYLPPGGPNVRMDSHVYPDYLVPSNYDSLLGKLIVWGEDRQAAIERMKRALDEMVVAGVPTTAPYHLLILDHEAFRAGDVDTGFIPKHGDDLILPPEAARKKPNVVVEAAKRAHSRSKASV*Prototheca moriformis (UTEX 1435) Heteromeric acetyl-CoA carboxylase BCCP-Asubunit allele 1 SEQ ID. NO: 95MSGTFYSSPAPGEPPFVKVGDSVKKGQTVCIIEAMKLMNEIEAESSGTIVKVVAEHGKPVTPGAPLFIIKPPrototheca moriformis (UTEX 1435) Heteromeric acetyl-CoA carboxylase BCCP-Asubunit allele 2 SEQ ID. NO: 96MPSFTKALPRLGGGLVPKPAVSVTDSGKSDLLMRFFESDWFDAWIALTYVALGSEGRSCVEMAGLRAVRRFGTDWGHPGEWVWLERRVGLLREVVWPGRSRQSDALGPCIGVGGRARAGFKIARVQLTPLGDDYVTHYKPTRPPSRPPPLTAGCGDLPVAAHPAVHAAAQQLARARPDRPLLALAAHCGQDVLAAPRHQPGQPHRHARHRAPRSMRAGRPGRHLGTRGEETEGGRTXXXXPAGGPCRPPAAPPPPAPAVEGVEISSPMSGTFYSSPAPGEPPFVKVGDSVKKGQTVCIIEAMKLMNEIEAEVGDSGVVAVKEGPrototheca moriformis (UTEX 1435) Acetyl-CoA carboxylase alpha-CTsubunit allele 1 SEQ ID. NO: 97MSDPVASGCDSMTSSQCRVGAWASPMAVARTRAVARPIPRSRNPRLRVRAGIDPPDLPKPREEEKKHGDWFQMLLSRFNPISEKATNATVLDFEKPLVELDHKIREVRKVAEENGVDVTEQIRELEDRAQQLRKETYSKLTPIQRLQVARHPNRPTELDIALNITDKFVELHGDRAGYDDPALVCGIASIDNVSFMFMGHQKGRNTKENIYRNEGMPQPNGYRKALRFMRLADKFGFPIVTFVDTPGAYAGLKAEELGQGEAIAVNLREMFGFRVPIISIVIGEGGSGGALAIGCANRMLIMENAVYYVASPEACAAILWKSRDKAGLATEALKITPADLVKLKVMDEIVPEPLGAAHTDPMGAFPAVREAILRHWRELQPLSPVQMRADRYMKERHIGVYAEHMVPGGQIEAVEAFRESLPGAKTKAGTYAPTQEEADYIEQMAERWRWWDSLLEDKKDIVNPPKTEHPGWLIPGFAEFAIGVAKAADARQARQGLEGESAAHANDAGIGTERDGSAEVNGANAKNGAHDESLAEAE*Prototheca moriformis (UTEX 1435) Acetyl-CoA carboxylase alpha-CT subunitallele 2 SEQ ID. NO: 98proteinMLLSRFNPISEKATNATVLDFEKPLVELDHKIREVRKVAEENGVDVTEQIRELEDRAQQVVESVWRGLRKETYSKLTPIQRLQVARHPNRPTELDIALNITDKFVELHGDRAGYDDPALVCGIASIDNVSFMFMGHQKGRNTKENIYRNFGMPQPNGYRKALRFMRLADKFGFPIVTFVDTPGAYAGLKAEELGQGEAIAVNLREMFGFRVPIISIVIGEGGSGGALAIGCANRMLIMENAVYYVASPEACAAILWKSRDKAGLATEALKITPADLVKLKVMDEIVPEPLGAAHTDPMGAFPAVREAILRHPrototheca moriformis (UTEX 1435) Acetyl-CoA carboxylase alpha-CT subunitallele 2 SEQ ID. NO: 99ATGCTGCTCTCGCGCTTCAACCCCATCAGCGAGAAGGCCACCAACGCCACGGTGCTCGACTTTGAGAAACCTTTGGTGGAACTTGATCATAAAATTCGCGAGGTCCGCAAAGTCGCGGAGGAGAACGGCGTGGACGTCACGGAGCAGATCCGCGAGCTGGAGGACCGCGCGCAGCAGGTAGTCGAGTCTGTGTGGAGAGGGTTGCGAAAGGAGACGTACTCCAAGCTGACGCCGATCCAGCGGCTGCAGGTGGCGCGCCACCCCAACCGCCCCACTTTCTTGGACATTGCGCTCAACATCACGGACAAGTTTGTGGAGCTGCACGGCGATCGCGCGGGGTACGACGACCCGGCGCTCGTCTGCGGCATCGCGTCGATCGACAACGTGAGCTTCATGTTCATGGGCCACCAGAAGGGCCGCAACACCAAGGAGAACATCTACCGCAATTTCGGCATGCCGCAGCCCAACGGCTACCGCAAGGCGCTGCGCTTCATGCGCCTGGCGGACAAGTTTGGCTTCCCCATCGTCACGTTTGTGGACACGCCCGGCGCGTACGCGGGGCTCAAGGCCGAGGAGCTGGGCCAGGGCGAGGCCATCGCGGTCAACCTGCGCGAGATGTTTGGCTTCCGCGTGCCCATCATCTCCATCGTCATCGGCGAGGGCGGCTCCGGCGGCGCGCTGGCCATCGGCTGCGCCAACCGCATGCTCATCATGGAGAACGCGGTGTACTACGTGGCCTCTCCCGAGGCCTGCGCGGCCATCCTGTGGAAGAGCCGCGACAAGGCGGGCCTGGCCACCGAGGCGCTCAAGATCACGCCCGCCGACCTGGTCAAGCTCAAGGTCATGGACGAGATCGTGCCGGAGCCGCTGGGCGCCGCGCACACCGACCCCATGGGCGCCTTCCCGGCCGTGCGCGAGGCCATCCTGCGCCACPrototheca moriformis (UTEX 1435) 1d-ACCase-CT-alpha-A SEQ ID. NO: 100ATGGATCTCTTCCGCAGCGAGGAGGTGCACCTGATGCAGCTCATGATCCCCGCCGAGGTCGCCCACGACACGATGGTGGCCCTGGGCGAGGTGGGGATGCTGCAGTTCAAGGACCTCAACCCCGACAAGCCGGCCTTCCAGCGCACGTTCGCCAACCAGATCAAGCGGTGCGACGAGATGGCGCGGCAGCTGCGCTTCTTCCAGGCGGAGCTGGACAAGCACGAGGTGCCCGCTTCGCGGCGCGCGGGCGACCCGCCCAGCCCCGACCTGGACCAGCTGGAGTCGCAGCTTGCGGCGCTGGAGTCGGAGCTGGTGCAGCTCAACGGCAACGCGGAGCGCCTGGGACTAGGCGGTCGGCCCGCGCCTGTCGTCCCTTTGGAGGAGGAGAAGAAGCACGGCGACTGGTTCCAAATGCTGCTCTCGCGCTTCAACCCCATCAGCGAGAAGGCCACCAACGCCACGGTGCTCGACTTTGAGAAACCTTTGGTGGAACTTGATCATAAAATTCGCGAGTTGCGAAAGGAGACATACTCCAAGCTGACGCCGATCCAGCGGCTGCAGGTGGCGCGCCACCCCAACCGCCCTACTTTCTTGGACATTGCGCTCAACATCACGGACAAGTTTGTGGAGCTGCACGGCGACCGCGCGGGGTACGACGACCCCGCGCTCGTCTGCGGCATCGCGTCGATCGACAACGTGAGCTTCATGTTCATGGGCCACCAGAAGGGCCGCAACACCAAGGAGAACATCTACCGAAACTTTGGCATGCCGCAGCCCAACGGCTACCGCAAGGCGCTGCGCTTCATGCGCCTGGCCGACAAGTTTGGCTTCCCCATCGTCACGTTTGTGGACACGCCCGGCGCGTACGCGGGGCTCAAGGCCGAGGAGCTGGGCCAGGGCGAGGCCATCGCGGTCAACCTGCGCGAGATGTTTGGCTTCCGCGTGCCCATCATCTCCATCGTCATCGGCGAGGGCGGCTCCGGCGGCGCGCTGGCCATCGGCTGCGCCAACCGCATGCTCATCATGGAGAACGCGGTGTACTACGTGGCGTCCCCGGAAGCCTGTGCGGCCATCCTGTGGAAGAGCCGCGACAAGGCGGGCCTGGCCACCGAGGCGCTCAAGATCACGCCCGCCGACCTGGTCAAGCTCAAGGTCATGGACGAGATCGTACCCGAGCCGCTGGGCGCCGCGCACACCGACCCCATGGGCGCCTTCCCGGCCGTGCGCGAGGCCATTCGCCAGAAAAACGTCGTGGAAAACTTTGAGGCGGCGCTGGAGTACAACCCCGAGGCGTTTGGCACCGTCACCATGCTGTACGTGCCCATGACGGTCAACGGCACGCCCCTCAAGGCCTTTATCGATTCGGGCGCACAGATGACCATCATGTCCCGCAGCTGCGCGGAGCGGTGCGGGCTGCTCCGTCTCATGGACACGCGGTGGCAGGGGACAGCCGTCGGCGTCGGCTCGGCCAAGATCCTGGGGAGGATCCACATGGCTCCGCTGGTCGCGGCCGGCCACCACCTGCCCATCTCCATCACCGTGCTGGATCAGGAGGGCATGGACTTTCTCTTTGGGCTGGACAACCTCAAGCGGCACCAATGCTGCATCGACCTGAAGGCAAACGCGCTTCGGTTTGGGTCGACAGAGGCCGAGATCCCGTTTCTGCCGGAGCACGAGGTCCCCAGGCATCAGCACCAGCTCGCCGGGGAGGACGCCGCGAGCGGTAGAACCGCAGCCACGGCCCCTCCCAGCACCGCTCCCGCACCGACCAACCAGCCCACAGTGCAAGCAGCTCCGCCAGCACCAAATGCTCAGCTGGAGGAAAAGGTGTCGAAACTCATGGGCCTGGGCGGCGTGATCCAGCTATGGGATTACCGCATGGGCACGCTCATCGATCGATTCGAGGAGCATGAGGGACCCGTGCGTGGCGTGCACTTTCATCCCACCCAGCCGCTCTTCGTGTCTGGCGGGGACGACTACAAGATCAAAGTGTGGAACTACAAGACGCGCPrototheca moriformis (UTEX 1435) Heteromeric acetyl-CoA carboxylase BCCP-Asubunit allele 1 SEQ ID. NO: 101ATGTCCGGCACTTTCTACTCCTCCCCCGCCCCCGGCGAGCCACCGTTTGTCAAGGTCGGCGACAGCGTCAAGAAGGGCCAGACGGTCTGCATCATCGAGGCCATGAAGCTGATGAACGAGATCGAGGCCGAGTCTTCTGGAACAATCGTCAAGGTTGTGGCCGAGCACGGCAAGCCTGTCACCCCCGGCGCCCCCCTGTTCATCATCAAGCCCTGAPrototheca moriformis (UTEX 1435) Heteromeric acetyl-CoA carboxylase BCCP-Asubunit allele 2 SEQ ID. NO: 102ATGCCGAGCTTTACCAAGGCGCTCCCCAGGCTGGGAGGGGGACTGGTCCCCAAGCCCGCAGTGAGCGTGACGGACAGCGGCAAGTCCGACTTGCTCATGCGTTTCTTTGAGAGCGATTGGTTCGACGCCTGGATTGCTCTTACGTACGTTGCGCTCGGTTCGGAGGGGCGTTCCTGTGTCGAGATGGCGGGGTTGCGAGCTGTGAGACGTTTTGGAACGGATTGGGGGCACCCTGGGGAGTGGGTTTGGCTGGAGCGGCGGGTGGGCTTGTTGAGGGAGGTGGTCTGGCCTGGTCGTTCAAGGCAGTCTGACGCATTGGGGCCGTGCATTGGCGTCGGGGGGCGTGCCCGCGCTGGTTTCAAAATCGCTCGCGTCCAATTGACGCCATTAGGTGATGATTATGTGACGCATTACAAACCCACCCGGCCCCCGTCGCGCCCGCCCCCGTTGACAGCGGGATGTGGAGACCTACCTGTCGCAGCTCACCCAGCTGTGCATGCTGCAGCCCAACAGCTCGCTCGAGCGCGTCCTGATCGACCTCTGCTCGCGCTCGCTGCGCATTGCGGTCAAGACGTACTGGCTGCTCCTCGCCATCAGCCAGGACAACCCCACAGACACGCACGTCATCGCGCTCCGCGATCGATGCGAGCAGGCCGCCCTGGAAGGCACCTGGGTACGAGGGGAGAGGAGACAGAGGGGGGGAGGACCNNNNNNNNNNCACCGGCCGGCGGGCCTTGTCGGCCTCCCGCCGCGCCGCCGCCGCCCGCCCCCGCCGTCGAGGGCGTGGAGATCAGCTCGCCCATGTCCGGCACCTTCTACTCCTCCCCCGCCCCCGGCGAGCCGCCGTTTGTCAAGGTCGGCGACAGCGTCAAGAAGGGCCAGACGGTCTGCATCATCGAGGCCATGAAGCTCATGAACGAGATCGAGGCCGAGGTGGGCGACAGCGGAGTGGTGGCGGTGAAAGAGGGGTAAPrototheca moriformis (UTEX 1435) Heteromeric acetyl-CoA carboxylase BCsubunit allele 2 SEQ ID. NO: 103ATGATCACNGGCGTGGACCTGATCCAGGAGCAGATCCGCGCCGCCATGGGCGAGCCGCTGCGCCTGAGACAGGAGGACATCGTGATCAAGGGCCACAGCATCGAGTGCCGCATCAACGCGGAGGACCCNTTCAAGGGCTTCCGCCCGGGGCCGGGGCGCGTGACCGCCTACCTGCCGCCCGGCGGGCCCAACGTGCGCATGGACAGCCACGTCTACCCCGACTACCTGGTGCCCTCCAACTACGACTCCCTGCTGGGCAAGCTCATCGTCTGGGGCGAGGACCGCCAGGCGGCCATCGAGCGCATGAAGCGCGCGCTGGACGAGATGPrototheca moriformis ACC-BC-1 nucleotide sequence SEQ ID. NO: 104ATGGCGCCAGCCTCTGTCAACCTGACCCAGGTTTGCTGCTCCGCCGGGCATGGTCTGCGTCCCGCGAGGGCGACGCCCTGCCCTGCGGCGGTGGCCGGCCGGAGGCAGTTCCCCCGCGGCGCCGGCCCCTCCCGTCGCTCCCAGCGCTCGACGGGCTCCGCGAGGTTGACGCCCTCTGCCTCCACGCCCTCCCGCCTGGTCGCCCGCGCCGAGACGGGCACCAATGGCTCCGCCGCCCTGACCCCCATCCACAAGGTGCTGATCGCCAACCGTGGCGAGATCGCGGTTCGCGTGATCCGGGCCTGCAAGGAGCTGGGCATCAAGACGGTGGCCGTCTACTCCACGGCCGACCGCGAGTGCCTGCACGCGCAGCTGGCGGACGAGGCGGTGTGCATCGGCGAGGCGCCCAGCTCCGAGTCCTACCTCAACATCCCGACCATCCTCTCGGCGGCCATGTCGCGCGGCGCGGACGCGATCCACCCCGGCTACGGCTTTCTGTCCGAGAACGCCGAGTTTGTGGACATCTGCAAGGACCACGGCATCAACTTCATCGGCCCCGGGTCGGAGCACATCCGCGTGATGGGCGACAAGGCCACCGCGCGCGAGACGATGAAGCAGGCGGGCGTGCCGACGGTGCCCGGCTCGGACGGGCTGGTGGAGACGCCCGAGGAGGCGCTGGAGGTGGCGGACCAGGTGGGCTTCCCCGTGATGATCAAGGCCACGGCCGGCGGCGGCGGGCGCGGCATGCGCCTGGCCATGACGCGCGACGAGTTTTTGCCGCTGCTGCGCGCGGCGCAGGGCGAGGCGCAGGCGGCCTTTGGCAACGGCGCCGTCTACCTGGAGCGCTACGTGAAGGACCCGCGGCACATCGAGTTCCAGGTGCTGGCGGACAAGCACGGCAACGTGATCCACCTGGGCGAGCGCGACTGCTCCATCCAGCGCCGCAACCAGAAGCTGCTGGAGGAGGCGCCCAGCCCCGCGCTCACGCCCGAGGTGCGCGCGGCCATGGGCGAGGCGGCCACCAACGCCGCGCGCAGCATCGGCTACGTGGGCGTGGGCACCATCGAGTTCCTGTGGGAGCCGGCCGGCTTCTACTTCATGGAGATGAACACGCGCATCCAGGTGGAGCACCCGGTGACGGAGATGATCACGGGCGTGGACCTGATCCAGGAGCAGATCCGCGCCGCCATGGGCGAGCCGCTGCGCCTGACGCAGGAGGACATCGTGATCAAGGGCCACAGCATCGAGTGCCGCATCAACGCGGAGGACCCGTTCAAGGGCTTCCGCCCGGGGCCGGGGCGCGTGACCGCCTACCTGCCGCCCGGCGGGCCCAACGTGCGCATGGACAGCCACGTCTACCCCGACTACCTGGTGCCCTCCAACTACGACTCCCTGCTGGGCAAGCTCATCGTCTGGGGCGAGGACCGCCAGGCGGCCATCGAGCGCATGAAGCGCGCGCTGGACGAGATGGTCGTCGCCGGCGTGCCCACCACCGCGCCCTACCACCTGCTCATCCTCGACCACGAGGCCTTCCGCGCGGGCGACGTCGACACGGGCTTCATCCCCAAGCACGGCGACGACCTCATCCTGCCCCCGGAGGCCGCGCGCAAGAAGCCAAACGTCGTCGTCGAGGCCGCCAAGCGCGCCCACAGCCGCAGCAAGGCCTCCGTCTGACodon-optimized Prototheca moriformis (UTEX 1435) KASII SEQ ID NO: 105ATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCGCCGCCGCCGCCGACGCCAACCCCGCCCGCCCCGAGCGCCGCGTGGTGATCACCGGCCAGGGCGTGGTGACCTCCCTGGGCCAGACCATCGAGCAGTTCTACTCCTCCCTGCTGGAGGGCGTGTCCGGCATCTCCCAGATCCAGAAGTTCGACACCACCGGCTACACCACCACCATCGCCGGCGAGATCAAGTCCCTGCAGCTGGACCCCTACGTGCCCAAGCGCTGGGCCAAGCGCGTGGACGACGTGATCAAGTACGTGTACATCGCCGGCAAGCAGGCCCTGGAGTCCGCCGGCCTGCCCATCGAGGCCGCCGGCCTGGCCGGCGCCGGCCTGGACCCCGCCCTGTGCGGCGTGCTGATCGGCACCGCCATGGCCGGCATGACCTCCTTCGCCGCCGGCGTGGAGGCCCTGACCCGCGGCGGCGTGCGCAAGATGAACCCCTTCTGCATCCCCTTCTCCATCTCCAACATGGGCGGCGCCATGCTGGCCATGGACATCGGCTTCATGGGCCCCAACTACTCCATCTCCACCGCCTGCGCCACCGGCAACTACTGCATCCTGGGCGCCGCCGACCACATCCGCCGCGGCGACGCCAACGTGATGCTGGCCGGCGGCGCCGACGCCGCCATCATCCCCTCCGGCATCGGCGGCTTCATCGCCTGCAAGGCCCTGTCCAAGCGCAACGACGAGCCCGAGCGCGCCTCCCGCCCCTGGGACGCCGACCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGTGCTGGTGCTGGAGGAGCTGGAGCACGCCAAGCGCCGCGGCGCCACCATCCTGGCCGAGCTGGTGGGCGGCGCCGCCACCTCCGACGCCCACCACATGACCGAGCCCGACCCCCAGGGCCGCGGCGTGCGCCTGTGCCTGGAGCGCGCCCTGGAGCGCGCCCGCCTGGCCCCCGAGCGCGTGGGCTACGTGAACGCCCACGGCACCTCCACCCCCGCCGGCGACGTGGCCGAGTACCGCGCCATCCGCGCCGTGATCCCCCAGGACTCCCTGCGCATCAACTCCACCAAGTCCATGATCGGCCACCTGCTGGGCGGCGCCGGCGCCGTGGAGGCCGTGGCCGCCATCCAGGCCCTGCGCACCGGCTGGCTGCACCCCAACCTGAACCTGGAGAACCCCGCCCCCGGCGTGGACCCCGTGGTGCTGGTGGGCCCCCGCAAGGAGCGCGCCGAGGACCTGGACGTGGTGCTGTCCAACTCCTTCGGCTTCGGCGGCCACAACTCCTGCGTGATCTTCCGCAAGTACGACGAGATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAPrototheca moriformis (UTEX 1435) KASII SEQ ID NO: 106MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAAAAADANPARPERRVVITGQGVVTSLGQTIEQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALESAGLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMDIGFMGPNYSISTACATGNYCILGAADHIRRGDANVMLAGGADAAIIPSGIGGFIACKALSKRNDEPERASRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATILAELVGGAATSDAHHMTEPDPQGRGVRLCLERALERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVIPQDSLRINSTKSMIGHLLGGAGAVEAVAAIQALRTGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLDVVLSNSFGFGGHNSCVIFRKYDEMDYKDHDGPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK1) allele 1SEQ ID NO: 107ATGGTTTTGAACGCGGGGAGTTCGTCCCTCAAGTTCAAGGTGTTTGACAAGATCGGGGGCAAGCTGCAGCCCCTGGCGTCCGGTCTGTGCGAGCGCATCGGCGACACCGCCAACTCCCGCATGAAGGCCTTTACGGAAAAGGACGGCAACGTGCTGGTGGAGGAGGGTTTCCAGGACCACCAGGACGCGCTCGGGGTCGTGTCGCGCTTTCTTGGTGATGCCTTTTCGTCCGGCTTCACGGAGCGCATGCACAGCGTGGGACACCGCGTGGTGCACGGCCTGACGATCAGCGAGCCCGTGCTGATCGACGAGGGCGTGCTCGCGACCATCCGCGAGGCGATCGACCTCGCGCCGCTGCACAACCCGGCCGGGCTGCAGGGCATCGAGGCGGCCATGCGCACCTTTGCCTCGGCGCCGCACGTGGCCGTGTTTGACACGGCCTTCCACCCGCTCTGCGCGCGCAACCTGGGGCTCTACCTGGCGGTGCGCGCGGACGGCCACGGGCGGCCGCAGTACCGCGTGTACTGCGCGGTCCACTCGGCGCGCGAGCAGGAGCGCGACCGCAAGGCCTGGCAGGCGCGGCAGGAGGCGGCGCGCGCCTCGGCCGACCTGGAGGCGCGGCGCGGCCAGGCGCTCGCGCGCCTGGCCGAGCTGGGCTGGGTCCTGCTCCTCCAGGTGCACGACGAGGTCATCCTGGAAGGGCCGCGCGAGTCCGCCGACGAGGCGCGCGAGCGCGTCGTTGCCTGCATGCGCAGCCCTTTCACGGGCGCCACCGCGCAGCCCCTGCTCGTCGACCTGGTCGTCGACGCCAAGCACGCCGACACCTGGTACGACGCCAAGTAGPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK1) allele 1,protein SEQ ID NO: 108MVLNAGSSSLKFKVFDKIGGKLQPLASGLCERIGDTANSRMKAFTEKDGNVLVEEGFQDHQDALGVVSRFLGDAFSSGFTERMHSVGHRVVHGLTISEPVLIDEGVLATIREAIDLAPLHNPAGLQGIEAAMRTFASAPHVAVEDTAFHPLCARNLGLYLAVRADGHGRPQYRVYCAVHSAREQERDRKAWQARQEAARASADLEARRGQALARLAELGWVLLLQVHDEVILEGPRESADEARERVVACMRSPFTGATAQPLLVDLVVDAKHADTWYDAKPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK1) allele 2SEQ ID NO: 109ATGAAGGTATTCGCAGATTTTGGGGGCGGACTTGGGGCTGGCGTGGGTTCGCCTTGCTCCGCCCATGGGGTTTGGGTGCAGATCACGATGAATGAGAGAGAGAGGGAGGAGAGTGGGGCACTGCGTGGTGGGTGGGGTTTGTTGGTCGCTCCTCTCCAGGTCTCCAGAAGACAAGGAGAAATGGAGGGCCTTTACGGAAAAGGACGGCAACGTGCTGGTGGAGGAGGGTTTCCAGGACCACCAGGACGCGCTCGGGGTCGTGTCGCGCTTTCTTGGCGATGCCTTTTCGTCCGGCTTCACGGAGCGCATGCACAGCGTGGGGCACCGCGTGGTGCACGGGCTGACGATCAGCGAGCCCGTGCTGATCGACGAGGGCGTGATCGCGACCATCCGCGAGGCGATCGACCTGGCGCCGCTGCACAACCCGGCCGGGCTGCAGGGCATCGAGGCGGCCATGCGCACCTTTGCCTCGGCGCCGCACGTSGCCCGTGGCGTGCGCCGCTACGGCTTCCACGGCTCCAGCTACGCGTACCTCGTCCCGCGCGCCGCGGCCGTGCTGGGCAAGCCCGCCGCCGAGCTGMMSCGCCAGGCGCTCAGGTCCGCCATCCAGCCCTTCCACACGGGGCCGTCGAAGGCTGGACGGTGGCGGGGCCGCTGCTTGTCGGCGCTGGTGCCGTCCTCTTCGTCGCTGCTGGTGCTGACCTCATCGGGCTGTCGCAGCAGGCTGGCGTTGCAGGCCGTGGGTTGCAGCTCCACGAGGACCCTGCCGTGGTGTGCGGGTGGGGGTGGGTGAGCGGGACAGCCGTGGTGAGCGGGCTGCGAATGCAGTTGCGCCAATCAGGCGCCTGAPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK1) allele 2,protein SEQ ID NO: 110MKVFADEGGGLGAGVGSPCSAHGVWVQITMNEREREESGALRGGWGLLVAPLQVSRRQGEMEGLYGKGRQRAGGGGFPGPPGRARGRVALSWRCLEVRLHGAHAQRGAPRGARADDQRARADRRGRDRDHPRGDRPGAAAQPGRAAGHRGGHAHLCLGAAR?PWRAPLRLPRLQLRVPRPARRGRAGQARRRA??PGAQVRHPALPHGAVEGWTVAGPLLVGAGAVLEVAAGADLIGLSQQAGVAGRGLQLHEDPAVVCGWGWVSGTAVVSGLRMQLRQSGAPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK2) SEQ ID NO: 111ATGGTTTTGAACGCGGGGAGTTCGTCCCTGAAGTTCAAGGTGTTTGACAAGATCGGAGGGAAGCTGCAGCCCCTGGCGTCCGGTCTGTGCGAGCGCATCGGCGACACCGCCAACTCCCGCATGAAGGCCTTTACGGAAAAGGACGGCAACGTGCTGGTGGAGGAGGGTTTCCAGGACCACCAGGACGCGCTCGGGGTCGTGTCGCGCTTTCTTGGTGATGCCTTTTCGTCCGGCTTCACGGAGCGCATGCACAGCGTGGGGCACCGCGTGGTGCACGGGCTGACGATCAGCGAGCCCGTGCTGATCGACGAGGGCGTGATCGCGACCATCCGCGAGGCGATCGACCTGGCGCCGCTGCACAACCCGGCCGGGCTGCAGGGCATCGAGGCGGCCATGCGCACCTTTGCCTCGGCGCCGCACGTCGCCGTGTTCGACACGGCTTTCCACCAGACCATGCCGCCCAGCGCCTTCATGTACGCGCTGCCCTACGAGCTGTACGAGGCGCAGGGCGTGCGCCGCTACGGCTTCCACGGCTCCAGCTACGCCTACCTCGTCCCGCGCGCGGCGGCCGCGCTGGGCAAGCCCGCCGCCGAGCTGAACGCGGTCGTCTGCCACCTGGGCGCCGGCGCCAGCATGGCCGCCATCCGCGGCGGGCGCTCGGTGGACACCACCATGGGCCTCACCCCGCTGGAGGGCCTKGTCATGGGCACGCGCTGCGGCGACATCGACCCCGCCATCGTGACCTACCTGCTGGGCAAGGGCCACTCGGCCAAGGACGTGGACGCGCTCATGAACAAGAAGAGCGGCTTYGCCGGSCTGGCMGGGCACGCCGACCTGCGCCTGGTCATCGAGGGCGCCGACAAGGGCGACGAGCGCTGCCGCGTCGCGCTGGAGGTCTGGCGCCACCGCATCCGCAAGTACATCGGSGCCTACGCGATGCAGCTGGGCGCGCCSCTGGACGCCATCGTCTTCTCCGCCGGCATCGGCGAGAACAGCTCCATCCTGCGCAAGTACCTGCTGGAGGACCTGGAGTGGTGGGGCATCGAGCTGGACGAGGGCAAGAACAGTCTCTGCGTGGACGGCGTCGCGGGCGACATCTCCCGCGAYGGCTCYAAGACCCGCCTGCTCGTCATCCCCACCGACGAGGAGCTSAGCATCGCCGAACAGAGCATCGACGTCGTCAACGGCCTGGCCGCCTGAPrototheca moriformis (UTEX 1435) Acetate kinase 1 (ACK2), proteinSEQ ID NO: 112MVLNAGSSSLKFKVFDKIGGKLQPLASGLCERIGDTANSRMKAFTEKDGNVLVEEGFQDHQDALGVVSRFLGDAFSSGFTERMHSVGHRVVHGLTISEPVLIDEGVIATIREAIDLAPLHNPAGLQGIEAAMRTFASAPHVAVFDTAFHQTMPPSAFMYALPYELYEAQGVRRYGFHGSSYAYLVPRAAAALGKPAAELNAVVCHLGAGASMAAIRGGRSVDTTMGLTPLEGLVMGTRCGDIDPAIVTYLLGKGHSAKDVDALMNKKSGFAGLAGHADLRLVIEGADKGDERCRVALEVWRHRIRKYIGAYAMQLGAPLDAIVFSAGIGENSSILRKYLLEDLEWWGIELDEGKNSLCVDGVAGDISRDGSKTRLLVIPTDEELSIAEQSIDVVNGLAAPrototheca moriformis Phosphate acetyltransferase allele 1SEQ ID NO: 113ATGAGTCTGTCGTCTTCTCGGAGTTTGCGGCGGACGCTGAGCCTGCTGAGGCCGGTGGCGGCGTCCACGCCCCTGTTTGCGCGCCACCTGTCGAGCCGCGGCATCCCGGACGCGGACACGGTCTACTGCACGGACGTCTCCGCGCAGCGCGGCAACTCGCCCATCCTGCTGGGCCTGTTTGAGTACTTCCAGCGCCAGTCGCCCGACGTGGGCTTCTTCCAGCCCATCGGCGCGGACCCGCTGAAGAGCTACAAGCCGGCCCCCGGCGCGCCCAAGCACATCGCGCTCATCCACCAGGCCATGGGCCTGAAGGACTCGCCCGAGTCGATGTACGGCGTGACCGAGGACGAGGCCAAGCGCCTGCTGACGGCGGGGCGCTACGACGAGCTGGTGGACCGCGTGTTCACCAAGTTCCAGGCGGTCAAGCGCAGCAAGGAGATCGTGGTGATCGAGGGCGCCACCGTGCAGGGCATCGGCAACCACAACGAGCTCAACGCGCGCCTGGCCTCCGAGCTGGACAGCGGCGTGCTGATGATCATGGACCTCAACAGCGACGCGCCGGGGAGCGGCGAGGACATCGCCGGGCGCGCGCTGCGGTACAAGCGCGAGTTCGAGGCGGAGCGCGCGCGCGTGAGCGGGCTCATCCTGAACAAGGTGCCCTTTGGCGCGGAGGAGGCGCTGATCGAGGCGGTCCGGCGCGAGCTGGAGGGCCGGGACCTGCCCTTTGCGGGCGGCCTGCCCTACAGCCGCGTCATCGGCACCGCACGCGTGAACGAGATCGTCGAGTCGCTGGGCGCCAAGCTGCTGTTCGGGCGGCAGGAGCTGATCGACAGCGACGTCACGGGGTACATGATCGCCGCGCAGGCCATCGACGCCTTTCTGACGAAACTAGAGACGCTGCGCTCGCAGCGCACGAGCGAGGGCCAGGACTTTTTCCGGCCGCTGGTGGTGACCAACAAGGACCGGCAGGACCTGATCCTGGGCCTGGCGGCCGCGTCCCTGGCGGGCACGGGCCCGCAGCTGGGCGGGCTCATCCTCGCGGACGCGGACCTGACCGACCTGACCCCCGAGACGCGCGCGATCGTTAGCAAGCTCAACCCGGACCTGCCCATGCTGGAGGTGCCCTACGGCACCTTTGAGACGGCGCGCCGCCTGACGCGCGTGACGCCGGGCATCCTGCCCAGCTCGGTGCGCAAGGTTCGCGAGGCCAAGGCGCTCTTCCACCGGTACGTGGACGCAGACGTCATCGCGGCCGGGCTGGTGCGGCCGCAGCGCCCCAGCCTGACCCCCAAGCGCTTCCTGTACCAGATCGAGGAGATCTGCAAGGGCGAGCTGCAGAACGTGGTGCTCCCCGAGTCGCACGACCACCGTGTGCTCACGGCCGCGGCCGAGGTCGCGGCCAAGGGCCTGGCGCGCGTGACGCTGCTGGGCAGCCCCGAGGAGGTGGAGTCGGCCGCGCGCCGCTTCAACGTCGACATCAGCAAGTGCGACGTGATCGACTACAAGCACTCGCCCGAGCTGGACCGGTACGCGGACTGGCTGGTGGAGAAGCGCAAGGCCAAGGGCCTGACCAAGGAGGGCGCGCTGGACCAGCTGCAGGACCTCAACATGTACGCCACCGTCATGGTGGCCGTCGGCGACGCGGACGGCATGGTGTCGGGCGCGACCTGCACCACGGCCAACACCATCCGGCCTGCGCTGCAGGTGCTCAAGACGCCCGACCGCAAGCTCGTCTCCTCCATCTTCTTCATGTGCCTGCCGGACAAGGTCCTCGTCTACGGCGACTGCGCGGTCAACGTGACGCCCGCGCCGGGCGAGCTGGCGCAGATCGCGACCGTGTCGGCCGACACGGCCGCCGCCTTTGGCATCGACCCGCGCGTGGCCATGCTCTCCTACTCGACCCTGGGCTCTGGCTCGGGGCCGCAGGTGGACATGGTCACCGAGGCCACGCGCCTCGCGCGCGAGGCGCGCCCCGACCTCAACATTGAGGGGCCCATCCAGTACGACGCCGCTGTCGACCCCGCCGTGGCCGCCACCAAGATCAAGGCGCACTCCGAGGTCGCGGGGCGCGCCTCCGTCTGCATCTTCCCGGATCTCAACACGGGGAACAACACCTACAAGGCCGTGCAGCAGTCCACGGGCGCCATCGCCATCGGACCGCTCATGCAGGGCCTCGCGCGCCCCGTCAACGATCTCTCCCGCGGCTGCACCGTCGCCGACATCGTCAACACCGTCGCCTGCACGGCCGTCCAGGCCATCGGGCTCAAAGCGCGCGACGCCGAGGCGGCGCGCGCGGGGGCCGCGGCTGCTGCTGCTTGAPrototheca moriformis Phosphate acetyltransferase, proteinSEQ ID NO: 114MSLSSSRSLRRTLSLLRPVAASTPLFARHLSSRGIPDADTVYCTDVSAQRGNSPILLGLFEYFQRQSPDVGFFQPIGADPLKSYKPAPGAPKHIALIHQAMGLKDSPESMYGVTEDEAKRLLTAGRYDELVDRVETKFQAVKRSKEIVVIEGATVQGIGNHNELNARLASELDSGVLMIMDLNSDAPGSGEDIAGRALRYKREFEAERARVSGLILNKVPFGAEEALIEAVRRELEGRDLPFAGGLPYSRVIGTARVNEIVESLGAKLLFGRQELIDSDVTGYMIAAQAIDAFLTKLETLRSQRTSEGQDFFRPLVVTNKDRQDLILGLAAASLAGTGPQLGGLILADADLTDLTPETRAIVSKLNPDLPMLEVPYGTFETARRLTRVTPGILPSSVRKVREAKALFHRYVDADVIAAGLVRPQRPSLTPKRFLYQIEEICKGELQNVVLPESHDHRVLTAAAEVAAKGLARVTLLGSPEEVESAARRFNVDISKCDVIDYKHSPELDRYADWLVEKRKAKGLTKEGALDQLQDLNMYATVMVAVGDADGMVSGATCTTANTIRPALQVLKTPDRKLVSSIFFMCLPDKVLVYGDCAVNVTPAPGELAQIATVSADTAAAFGIDPRVAMLSYSTLGSGSGPQVDMVTEATRLAREARPDLNIEGPIQYDAAVDPAVAATKIKAHSEVAGRASVCIFPDLNTGNNTYKAVQQSTGAIAIGPLMQGLARPVNDLSRGCTVADIVNTVACTAVQAIGLKARDAEAARAGAAAAAA*Prototheca moriformis (UTEX 1435) Phosphate acetyltransferase allele 2SEQ ID NO: 115ATGGTCAACGACTTGGTGGGAGCCATGGCGATGGTCGGCGACGGCAAGCACGGGCGCAGGGCAGCACGGGGCGGTCAGCGAGGGGCTTGGGTGCAACTGTGTGGGTCGTCAGCAGGCCCTAATCAATCCAGGGACCCTTCTTCAGCGCAATTGGCCTCGTTCTATCGCGATCGCGATGACATATACTCTCGTCACCTCAAGGGGTTGAGCGAGTTCGCATTCATAAGTCAGTGGCATCGGTCTTGCCGGGTTTGGGCATTGTGCCTGGGTCTGATCTGGAAACCCGACTCGCCGAAGGTGCCCATCGGCCGGTCATCGCGGGACCCCCCCACCCCCGCCGGCCTCGTTGATTGGCAGCATGAGTCTGTCGTCTTCTCGGAGTTTGCGGCGGACGCTGAGCCTGCTGAGGTGCGATGCAAGGGCGTCCCTGGGCGATTTGACGGGCGGTTACCTAAGGCGGAGCGACGTGCGGCTCCGGCGGGCAGCCGACACGTGGGCTTGTTGCCATGGACAGGCCCAGATCCCGATCCGATGGGCCCTGTAACGTTTGCTTATTTGCCGACGCCCTTTGCCACGGCCTGCCCGCCAGAAATGCCGGGCAGCGCCCCGTTGCGCCGCTTCCCCATGCTTCTCGCCTCGAATCACATTTCGACACTCCTCCGACGTCTCTCCACCCCTCCCCGAATCACGCAGGCCAGTGGCGGCGTCCACGCACCTGTTTGCGCGCCACCTGTCGAGCCGCGGCATCCCGGACGCGGACACGGTCTACTGCACGGACGTCTCCGCGCAGCGCGGCAACTCGCCCATCCTGCTGGGCCTGTTCGAGTACTTCCAGCGCCAGTCGCCCGACGTGGGCTTCTTCCAGCCCATCGGCGCGGACCCGCTGAGGAGCTACAAGCCTGCCCCCGGCGCGCCCAAGCACATCGCGCTCATCCACCAGGCCATGGGCCTGAAGGACTCGCCCGAGTCGATGTACGGCGTGACCGAGGACGAGGCCAAGCGCCTGCTGACGGCGGGGCGCTACGACGAGCTGGTGGACCGCGTGTTCACCAAGTTCCAGGCGGTCAAACGCAGCAAGGAGATTGTGGTGATCGAGGGCGCCACCGTGCAGGGCATCGGCAACCACAACGAGCTCAACGCGCGCCTGGCCTCCGAGCTGGACAGCGGCGTGCTGATGATCATGGACCTSAACMGSGACGCSCCSGGSAGCGGCGAGGACATCGCCGGGCGCGCGCTGCGGTACAAGCGCGAGTTCGAGGCGGAGCGCGCGCGCGTGAGCGGGCTCATCCTGAACAAGGTGCCCTTTGGCGCGGAGGAGGCGCTGATCGAGGCGGTCCGGCGCGAGCTGGAGGGCCGGGACCTGCCCTTTGCGGGCGGCCTGCCCTACAGCCGCGTCATCGGCACCGCGCGCGTGAACGAGATCGTCGAGTCGCTGGGCGCCAAGCTGCTGTTCGGGCGGCASGAGCTGATCGACAGCGACGTCACGGGGTACATGATCGCCGCGCAGGCCATCGACGCSTTYCTGACSAARCTRGAGACGCTGCGCTCGCAGCGCACGAGCGAGGGCCAGGACTTTTTCCGGCCGCTGGTGGTGACCAACAAGGACCGGCAGGACCTGATCCTGGGCCTGGCGGCCGCGTCSCTGGCGGGCACGGGCCCGCAGCTGGGCGGGCTCATCCTSGCSGACGCGGACCTGACCGACCTGACCCCCGAGACGCGCGCGATCGTTAGPrototheca moriformis (UTEX 1435) Phosphate acetyltransferase allele 2,protein SEQ ID NO: 116MVNDLVGAMAMVGDGKHGRRAARGGQRGAWVQLCGSSAGPNQSRDPSSAQLASFYRDRDDIYSRHLKGLSEFAFISQWHRSCRVWALCLGLIWKPDSPKVPIGRSSRDPPTPAGLVDWQHESVVESEFAADAEPAEVRCKGVPGREDGRLPKAERRAAPAGSRHVGLLPWTGPDPDPMGPVTFAYLPTPFATACPPEMPGSAPLRREPMLLASNHISTLLRRLSTPPRITQASGGVHAPVCAPPVEPRHPGRGHGLLHGRLRAARQLAHPAGPVRVLPAPVARRGLLPAHRRGPAEELQACPRRAQAHRAHPPGHGPEGLARVDVRRDRGRGQAPADGGALRRAGGPRVHQVPGGQTQQGDCGDRGRHRAGHRQPQRAQRAPGLRAGQRRADDHGP Prototheca moriformis Lactate dehydrogenaseSEQ ID NO: 117ATGGCTGAGGGGAGCATCGACTACAAGGTGGCTGTCTTCAGCTCCAGCGAGTGGGTGACCGACTCGTTCGCCGAGCCCCTCAAGATCTTCAAGGAGGTCTCCTACCTGCCGGCCCGCCTGGAGCCGACCAGCGCTGCCCTGGCGCACGGCTTTGACGCGGTGTGCATCTTTGTGAACGACGACGCTGGCGCGGAGACGCTCAAGGTTCTGGCGGAGGGCGGCGTGAAGCTGATCGCCATGCGCTGCGCAGGGTACGACCGCGTGGACCTGGCCGCGGCCAAGGAGCTGGGCATCCGCGTGGTGCGCGTGCCGGCCTACAGTCCGCGCTCGGTGGCGGAGCACGCGCTGGCGCTGATGATGGCGCTGGCGCGCAACATCAAGGCCTCGCAGATCAAGATGGCGGCCGGGTCCTACACGCTCAACGGCCTGGTGGGCGTGGAGCTGACGGGCAAGACCTTTGGCGTGGTGGGCACGGGCGCCATCGGGCGCGAGTTCGTCAAGCTGCTCAAGGGCTTTGAGGGCAAGGTGCTGGGCTACGACCCCTACCCGACCGACGCGGCGCGCGAGCTGGGCGTGGAGTACGTCGACCTGGACACGCTGCTGGCGCAGTCGGACGTCATCTCGCTGCACGTGCCGCTGCTGCCCAGCACGCAGAAGCTGCTGGGCACCGAGTCGCTGAGCAAGCTCAAGCCCACCGCGCTGGTCATCAACGTCAGCCGCGGCGGGCTCATCGACACCGAGGCCTGCATCCAGGCGCTGCAGGAGGACAAGTTTGCGGGCCTGGCCGTCGACGTCTACGAGGGCGAGGGCGCGCTCTTCTTCCAGGACTGGGCCAACATGAACGCCGCGCGCCGCATGAAGCAGTGGGACCAGAAGTTCATCGCGCTCAAGTCCATGCCCCAGGTCATCGTCACGCCGCACATCGCCTTCCTCACGCACAGCGCGCTCGACGCCATCGCCGCCACCACGCGCGACAACCTGCTCGCCGCCGCGCAGGGCAAGGACCTCGTCAACGAGGTCAAPrototheca moriformis lactate dehydrogenase protein SEQ ID NO: 118MAEGSIDYKVAVFSSSEWVTDSFAEPLKIFKEVSYLPARLEPTSAALAHGFDAVCIFVNDDAGAETLKVLAEGGVKLIAMRCAGYDRVDLAAAKELGIRVVRVPAYSPRSVAEHALALMMALARNIKASQIKMAAGSYTLNGLVGVELTGKTFGVVGTGAIGREFVKLLKGFEGKVLGYDPYPTDAARELGVEYVDLDTLLAQSDVISLHVPLLPSTQKLLGTESLSKLKPTALVINVSRGGLIDTEACIQALQEDKFAGLAVDVYEGEGALFFQDWANMNAARRMKQWDQKFIALKSMPQVIVTPHIAFLTHSALDAIAATTRDNLLAAAQGKDLVNEVPrototheca moriformis (UTEX 1435) LDP1 SEQ ID NO: 119ATGGCNGCTGTCGCTGAGAACCCCGCTCCCCCCCGCCCGAACAGCCTTAAGAAGCTGGGCTTCGTTCCCGAGGCCGGCGCGAAGAGCTACGAGATTGCCACCAATGTCTACACTACGGCAAAGAGCTACGTTCCCGCTAGCCTGCAGCCCAAGCTGGAGAAGGTGGAGGAGTCTGTGACCAGCGTGAGCGCCCCGTACGTGGCCAAGGCCCAGGACAAGACCGCGACCCTGCTCAAGGTGGCCGACGAGAAGGTTGATGAGGCCTTCGCCAAGGCCACCCAGGTCTACCTTAACAACAGCAAGTACGTCGCCGACACCCTGGACAAGCAGCGCGCCTACCACGCGCAGAACCTCGAGTCCTACAAGGCCGCGCGCGAGCACTACCTCAAGGTCGTCGAAAGCGGCGTCGAGTACGTGAAGAAGAACGGCATCTCCGGCACCGCCAAGGCCGCCGCCGACGAGGTGGCCGCGCGCGTGAGCGAGGCGCGCGCCGTCCCCGGCGCCCTGCTGCACCGCGTGCAGGAGGCCGTGGACAAGCTGCTGGCCAGCACGCCCGTGCACGGCACGGTCGAGCGCCTGAAGCCGGCCCTGGACAGCGCCTACCAGCGCTACAGCTCCCTGCACGACAACGTCGTCTCCAGCGACCAGTACCGCAAGGTCGTGAAGACCGGCTCGGACGTGCTCGCCCGCGTCGAGGCCTCGCCCATCTTCGCCAAGTCCAAGGCGACCCTGTACCCCTACGTCGCGCCCTACGCGGAGCCCGCGGCAGCGCGCCTGCAGCCCTACTACCTCCGGGTGGTCGAGCACCTCGCGCCCAAGGACGCCTGAATTTGTGGACGCGGCGGTCGCACTGCTGCCGCGTTGCGCGGTCTCGCTTGATCGCGGGGCTGCCCCCTTGGAGCGCTTGTTCGCGCCGGCGCGACGCGCCGTCCCAGTTGATTGTCGGGGCGGGGCGCCGTCACCGCGCGTGGGCCATCGGAATTGGATCTGCTGAGAATGCAAAGGGCGACCGGTGAPrototheca moriformis (UTEX 1435) LDP1 protein SEQ ID NO: 120MAAVAENPAPPRPNSLKKLGFVPEAGAKSYEIATNVYTTAKSYVPASLQPKLEKVEESVTSVSAPYVAKAQDKTATLLKVADEKVDEAFAKATQVYLNNSKYVADTLDKQRAYHAQNLESYKAAREHYLKVVESGVEYVKKNGISGTAKAAADEVAARVSEARAVPGALLHRVQEAVDKLLASTPVHGTVERLKPALDSAYQRYSSLHDNVVSSDQYRKVVKTGSDVLARVEASPIFAKSKATLYPYVAPYAEPAAARLQPYYLRVVEHLAPKDA 6S 5′genomic donor sequence SEQ ID NO: 121GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACC 6S 3′genomic donor sequence SEQ ID NO: 122GAGCTCCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAGCS. cereviseae invertase protein sequence SEQ ID NO: 123MLLQAFLFLLAGFAAKISASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVATWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIETYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGTAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGKSKVKFVKENPYFTNRMSTONQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVKS. cereviseae invertase protein coding sequence codon optimized forexpression in P. moriformis {UTEX 1435) SEQ ID NO: 124ATGctgctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAChlamydomonas reinhardtii TUB 2 (B-tub) promoter/5′ UTR SEQ ID NO: 125CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC Chlorella vulgaris nitrate reductase 37UTR SEQ ID NO: 126GCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTNucleotide sequence of the codon-optimized expression cassette of S.cerevisiae suc2 gene with C. reinhardtii (J-tubulin promoter/5′UTR and C.vulgaris nitrate reductase 3′ UTR SEQ ID NO: 127CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCC Prototheca moriformis (UTEX 1435) Amt03 promoter SEQ ID NO: 128GGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCChlorella protothecoides (UTEX 250) stearoyl ACP desaturase transit peptidecDNA sequence codon optimized for expression in P. moriformis.SEQ ID NO: 129ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCC SEQ ID NO: 130ATGGTGGTGGCCGCCGCCGCCAGCAGCGCCTTCTTCCCCGTGCCCGCCCCCCGCCCCACCCCCAAGCCCGGCAAGTTCGGCAACTGGCCCAGCAGCCTGAGCCAGCCCTTCAAGCCCAAGAGCAACCCCAACGGCCGCTTCCAGGTGAAGGCCAACGTGAGCCCCCACGGGCGCGCCCCCAAGGCCAACGGCAGCGCCGTGAGCCTGAAGTCCGGCAGCCTGAACACCCTGGAGGACCCCCCCAGCAGCCCCCCCCCCCGCACCTTCCTGAACCAGCTGCCCGACTGGAGCCGCCTGCGCACCGCCATCACCACCGTGTTCGTGGCCGCCGAGAAGCAGTTCACCCGCCTGGACCGCAAGAGCAAGCGCCCCGACATGCTGGTGGACTGGTTCGGCAGCGAGACCATCGTGCAGGACGGCCTGGTGTTCCGCGAGCGCTTCAGCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCAGCATCGAGACCCTGATGAACCACCTGCAGGACACCAGCCTGAACCACTGCAAGAGCGTGGGCCTGCTGAACGACGGCTTCGGCCGCACCCCCGAGATGTGCACCCGCGACCTGATCTGGGTGCTGACCAAGATGCAGATCGTGGTGAACCGCTACCCCACCTGGGGCGACACCGTGGAGATCAACAGCTGGTTCAGCCAGAGCGGCAAGATCGGCATGGGCCGCGAGTGGCTGATCAGCGACTGCAACACCGGCGAGATCCTGGTGCGCGCCACCAGCGCCTGGGCCATGATGAACCAGAAGACCCGCCGCTTCAGCAAGCTGCCCTGCGAGGTGCGCCAGGAGATCGCCCCCCACTTCGTGGACGCCCCCCCCGTGATCGAGGACAACGACCGCAAGCTGCACAAGTTCGACGTGAAGACCGGCGACAGCATCTGCAAGGGCCTGACCCCCGGCTGGAACGACTTCGACGTGAACCAGCACGTGAGCAACGTGAAGTACATCGGCTGGATTCTGGAGAGCATGCCCACCGAGGTGCTGGAGACCCAGGAGCTGTGCAGCCTGACCCTGGAGTACCGCCGCGAGTGCGGCCGCGAGAGCGTGGTGGAGAGCGTGACCAGCATGAACCCCAGCAAGGTGGGCGACCGCAGCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCGCCGACATCATGAAGGGCCGCACCGAGTGGCGCCCCAAGAACGCCGGCACCAACCGCGCCATCAGCACCTGACuphea wrightii FatB2 thioesterase; Gen Bank Accession No. U56104SEQ ID NO: 131MVVAAAASSAFFPVPAPRPTPKPGKFGNWPSSLSQPFKPKSNPNGRFQVKANVSPHPKANGSAVSLKSGSLNTLEDPPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLDRKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCKSVGLLNDGFGRTPEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGREWLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLHKFDVKTGDSICKGLTPGWNDFDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRECGRESVVESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNRAIST pSZ1500 SEQ ID NO: 137GGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGACCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCACTCCATAAACTCAAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGGGTACCCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATCAATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCCTAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTCTAAAGAACTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCGCCACCGGCGAGCAGCCCTCCGGCGTGGCCTCCCTGCGCGAGGCCGACAAGGAGAAGTCCCTGGGCAACCGCCTGCGCCTGGGCTCCCTGACCGAGGACGGCCTGTCCTACAAGGAGAAGTTCGTGATCCGCTGCTACGAGGTGGGCATCAACAAGACCGCCACCATCGAGACCATCGCCAACCTGCTGCAGGAGGTGGGCGGCAACCACGCCCAGGGCGTGGGCTTCTCCACCGACGGCTTCGCCACCACCACCACCATGCGCAAGCTGCACCTGATCTGGGTGACCGCCCGCATGCACATCGAGATCTACCGCTACCCCGCCTGGTCCGACGTGATCGAGATCGAGACCTGGGTGCAGGGCGAGGGCAAGGTGGGCACCCGCCGCGACTGGATCCTGAAGGACTACGCCAACGGCGAGGTGATCGGCCGCGCCACCTCCAAGTGGGTGATGATGAACGAGGACACCCGCCGCCTGCAGAAGGTGTCCGACGACGTGCGCGAGGAGTACCTGGTGTTCTGCCCCCGCACCCTGCGCCTGGCCTTCCCCGAGGAGAACAACAACTCCATGAAGAAGATCCCCAAGCTGGAGGACCCCGCCGAGTACTCCCGCCTGGGCCTGGTGCCCCGCCGCTCCGACCTGGACATGAACAAGCACGTGAACAACGTGACCTACATCGGCTGGGCCCTGGAGTCCATCCCCCCCGAGATCATCGACACCCACGAGCTGCAGGCCATCACCCTGGACTACCGCCGCGAGTGCCAGCGCGACGACATCGTGGACTCCCTGACCTCCCGCGAGCCCCTGGGCAACGCCGCCGGCGTGAAGTTCAAGGAGATCAACGGCTCCGTGTCCCCCAAGAAGGACGAGCAGGACCTGTCCCGCTTCATGCACCTGCTGCGCTCCGCCGGCTCCGGCCTGGAGATCAACCGCTGCCGCACCGAGTGGCGCAAGAAGCCCGCCAAGCGCATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAATCGATAGATCTCTTAAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTAATTAAGAGCTCCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGTGAGTGA 5′ FADc genomic region donor DNA SEQ ID NO: 138GGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGACCCATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGTTTGCGCACTCCATAAACTCAAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTGCTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGTTCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGCATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGCACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAGTGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCTGCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCG 3′FADc genomic region donor DNA SEQ ID NO: 139CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGTGAGTGA 5′donor DNA sequence of Prototheca moriformis FATA1 knockout homologousrecombination targeting construct SEQ ID NO: 140GCTCTTCGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGGGTACC 3′donor DNA sequence of Prototheca moriformis FATA1 knockout homologousrecombination targeting construct SEQ ID NO: 141GACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCTGGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCAATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCCCCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTATTCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCGGAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAATGGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTGAAGAGC Chlorella protothecoides actin promoter/5′UTR SEQ ID NO: 142ACTAGAGAGTTTAGGTCCAGCGTCCGTGGGGGGGGACGGGCTGGGAGCTTGGGCCGGGAAGGGCAAGACGATGCAGTCCCTCTGGGGAGTCACAGCCGACTGTGTGTGTTGCACTGTGCGGCCCGCAGCACTCACACGCAAAATGCCTGGCCGACAGGCAGGCCCTGTCCAGTGCAACATCCACGGTCCCTCTCATCAGGCTCACCTTGCTCATTGACATAACGGAATGCGTACCGCTCTTTCAGATCTGTCCATCCAGAGAGGGGAGCAGGCTCCCCACCGACGCTGTCAAACTTGCTTCCTGCCCAACCGAAAACATTATTGTTTGAGGGGGGGGGGGGGGGGGCAGATTGCATGGCGGGATATCTCGTGAGGAACATCACTGGGACACTGTGGAACACAGTGAGTGCAGTATGCAGAGCATGTATGCTAGGGGTCAGCGCAGGAAGGGGGCCTTTCCCAGTCTCCCATGCCACTGCACCGTATCCACGACTCACCAGGACCAGCTTCTTGATCGGCTTCCGCTCCCGTGGACACCAGTGTGTAGCCTCTGGACTCCAGGTATGCGTGCACCGCAAAGGCCAGCCGATCGTGCCGATTCCTGGGGTGGAGGATATGAGTCAGCCAACTTGGGGCTCAGAGTGCACACTGGGGCACGATACGAAACAACATCTACACCGTGTCCTCCATGCTGACACACCACAGCTTCGCTCCACCTGAATGTGGGCGCATGGGCCCGAATCACAGCCAATGTCGCTGCTGCCATAATGTGATCCAGACCCTCTCCGCCCAGATGCCGAGCGGATCGTGGGCGCTGAATAGATTCCTGTTTCGATCACTGTTTGGGTCCTTTCCTTTTCGTCTCGGATGCGCGTCTCGAAACAGGCTGCGTCGGGCTTTCGGATCCCTTTTGCTCCCTCCGTCACCATCCTGCGCGCGGGCAAGTTGCTTGACCCTGGGCTGGTACCAGGGTTGGAGGGTATTACCGCGTCAGGCCATTCCCAGCCCGGATTCAATTCAAAGTCTGGGCCACCACCCTCCGCCGCTCTGTCTGATCACTCCACATTCGTGCATACACTACGTTCAAGTCCTGATCCAGGCGTGTCTCGGGACAAGGTGTGCTTGAGTTTGAATCTCAAGGACCCACTCCAGCACAGCTGCTGGTTGACCCCGCCCTCGCAAAACGCCTTTGTACAACTGCAAtTHIC expression cassette comprising Chlorella protothecoides actinpromoter/5′UTR, Arabidopsis thaliana THIC protein coding sequence codon-optimized for expression in Prototheca moriformis, and Chlorella vulgarisnitrate reductase 3′ UTR SEQ ID NO: 143agtttaggtccagcgtccgtggggggggacgggctgggagcttgggccgggaagggcaagacgatgcagtccctctggggagtcacagccgactgtgtgtgttgcactgtgcggcccgcagcactcacacgcaaaatgcctggccgacaggcaggccctgtccagtgcaacatccacggtccctctcatcaggctcaccttgctcattgacataacggaatgcgtaccgctctttcagatctgtccatccagagaggggagcaggctccccaccgacgctgtcaaacttgcttcctgcccaaccgaaaacattattgtttgagggggggggggggggggcagattgcatggcgggatatctcgtgaggaacatcactgggacactgtggaacacagtgagtgcagtatgcagagcatgtatgctaggggtcagcgcaggaagggggcctttcccagtctcccatgccactgcaccgtatccacgactcaccaggaccagcttcttgatcggcttccgctcccgtggacaccagtgtgtagcctctggactccaggtatgcgtgcaccgcaaaggccagccgatcgtgccgattcctggggtggaggatatgagtcagccaacttggggctcagagtgcacactggggcacgatacgaaacaacatctacaccgtgtcctccatgctgacacaccacagcttcgctccacctgaatgtgggcgcatgggcccgaatcacagccaatgtcgctgctgccataatgtgatccagaccctctccgcccagatgccgagcggatcgtgggcgctgaatagattcctgtttcgatcactgtttgggtcctttccttttcgtctcggatgcgcgtctcgaaacaggctgcgtcgggctttcggatcccttttgctccctccgtcaccatcctgcgcgcgggcaagttgcttgaccctgggctgtaccagggttggagggtattaccgcgtcaggccattcccagcccggattcaattcaaagtctgggccaccaccctccgccgctctgtctgatcactccacactcgcgcacacactacgtccaagtcctgatccaggcgtgtctcgggacaaggtgtgctcgagtttgaatctcaaggacccactccagcacagctgctggCtgaccccgccctcgcaatctagaATGgccgcgtccgtccactgcaccctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgccaacgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcccttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgagacgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggacatccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattggcagcagcagctcggatagtatcgacacactcCggacgctggtcgtgtgatggactgttgccgccacacttgcCgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatcccctcccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcacCgcccctcgcacagccttggCttgggctccgcctgtattcCcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatccCodon-optimized sequence of PmKASII comprising a C. protothecoides S106stearoyl-ACP desaturase transit peptide SEQ ID NO: 144ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccgccgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagaccatcgagcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacaccaccaccatcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtgatcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccggcctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgacccgcggcggcgtgcgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatcggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgccgcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaaggccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagggcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcgccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccgcgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccggcgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccggcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttcggcggccacaactcctgcgtgatcttccgcaagtacgacgagatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGACodon-optimized amino acid sequence of PmKASII comprising a C.protothecoides S106 stearoyl-ACP desaturase transit peptideSEQ ID NO: 145MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAAAAADANPARPERRVVITGQGVVTSLGQTIEQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALESAGLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMDIGFMGPNYSISTACATGNYCILGAADHIRRGDANVMLAGGADAAIIPSGIGGFIACKALSKRNDEPERASRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATILAELVGGAATSDAHHMTEPDPQGRGVRLCLERALERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVIPQDSLRINSTKSMIGHLLGGAGAVEAVAAIQALRTGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLDVVLSNSFGFGGHNSCVIFRKYDEMDYKDHDGDYKDHDIDYKDDDDKCodon-optimized Protetheca moriformis (UTEX 1435) FAD2 protein-codingsequence SEQ ID NO: 146ATGgccatcaagaccaaccgccagcccgtggagaagccccccttcaccatcggcaccctgcgcaaggccatccccgcccactgcttcgagcgctccgccctgcgctcctccatgtacctggccttcgacatcgccgtgatgtccctgctgtacgtggcctccacctacatcgaccccgcccccgtgcccacctgggtgaagtacggcgtgatgtggcccctgtactggttcttccagggcgccttcggcaccggcgtgtgggtgtgcgcccacgagtgcggccaccaggccttctcctcctcccaggccatcaacgacggcgtgggcctggtgttccactccctgctgctggtgccctactactcctggaagcactcccaccgccgccaccactccaacaccggctgcctggacaaggacgaggtgttcgtgcccccccaccgcgccgtggcccacgagggcctggagtgggaggagtggctgcccatccgcatgggcaaggtgctggtgaccctgaccctgggctggcccctgtacctgatgttcaacgtggcctcccgcccctacccccgcttcgccaaccacttcgacccctggtcccccatcttctccaagcgcgagcgcatcgaggtggtgatctccgacctggccctggtggccgtgctgtccggcctgtccgtgctgggccgcaccatgggctgggcctggctggtgaagacctacgtggtgccctacctgatcgtgaacatgtggctggtgctgatcaccctgctgcagcacacccaccccgccctgccccactacttcgagaaggactgggactggctgcgcggcgccatggccaccgtggaccgctccatgggcccccccttcatggacaacatcctgcaccacatctccgacacccacgtgctgcaccacctgttctccaccatcccccactaccacgccgaggaggcctccgccgccatccgccccatcctgggcaagtactaccagtccgactcccgctgggtgggccgcgccctgtgggaggactggcgcgactgccgctacgtggtgcccgacgcccccgaggacgactccgccctgtggttccacaagTAGCodon-optimized Protetheca moriformis (UTEX 1435) amino acid sequence ofPrototheca moriformis FAD2 SEQ ID NO: 147MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGVMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVFHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVFVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMFNVASRPYPRFANHFDPWSPIFSKRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYLIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDNILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHKCodon-optimized coding region of Brassica napus C18:0-preferringthioesterase from pSZ1358 SEQ ID NO: 148ACTAGTATGCTGAAGCTGTCCTGCAACGTGACCAACAACCTGCACACCTTCTCCTTCTTCTCCGACTCCTCCCTGTTCATCCCCGTGAACCGCCGCACCATCGCCGTGTCCTCCGGGCGCGCCTCCCAGCTGCGCAAGCCCGCCCTGGACCCCCTGCGCGCCGTGATCTCCGCCGACCAGGGCTCCATCTCCCCCGTGAACTCCTGCACCCCCGCCGACCGCCTGCGCGCCGGCCGCCTGATGGAGGACGGCTACTCCTACAAGGAGAAGTTCATCGTGCGCTCCTACGAGGTGGGCATCAACAAGACCGCCACCGTGGAGACCATCGCCAACCTGCTGCAGGAGGTGGCCTGCAACCACGTGCAGAAGTGCGGCTTCTCCACCGACGGCTTCGCCACCACCCTGACCATGCGCAAGCTGCACCTGATCTGGGTGACCGCCCGCATGCACATCGAGATCTACAAGTACCCCGCCTGGTCCGACGTGGTGGAGATCGAGACCTGGTGCCAGTCCGAGGGCCGCATCGGCACCCGCCGCGACTGGATCCTGCGCGACTCCGCCACCAACGAGGTGATCGGCCGCGCCACCTCCAAGTGGGTGATGATGAACCAGGACACCCGCCGCCTGCAGCGCGTGACCGACGAGGTGCGCGACGAGTACCTGGTGTTCTGCCCCCGCGAGCCCCGCCTGGCCTTCCCCGAGGAGAACAACTCCTCCCTGAAGAAGATCCCCAAGCTGGAGGACCCCGCCCAGTACTCCATGCTGGAGCTGAAGCCCCGCCGCGCCGACCTGGACATGAACCAGCACGTGAACAACGTGACCTACATCGGCTGGGTGCTGGAGTCCATCCCCCAGGAGATCATCGACACCCACGAGCTGCAGGTGATCACCCTGGACTACCGCCGCGAGTGCCAGCAGGACGACATCGTGGACTCCCTGACCACCTCCGAGATCCCCGACGACCCCATCTCCAAGTTCACCGGCACCAACGGCTCCGCCATGTCCTCCATCCAGGGCCACAACGAGTCCCAGTTCCTGCACATGCTGCGCCTGTCCGAGAACGGCCAGGAGATCAACCGCGGCCGCACCCAGTGGCGCAAGAAGTCCTCCCGCATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAATCGATAmino acid sequence of Brassica napus C18:0-preferring thioesterase(Accession No. CAA52070.1) SEQ ID NO: 149MLKLSCNVTNNLHTFSFFSDSSLFIPVNRRTIAVSSSQLRKPALDPLRAVISADQGSISPVNSCTPADRLRAGRLMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGESTDGFATTLTMRKLHLIWVTARMHIETYKYPAWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDTRRLQRVTDEVRDEYLVFCPREPRLAFPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIGWVLESIPQEIIDTHELQVITLDYRRECQQDDIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQFLHMLRLSENGQEINRGRTQWRKKSSRPrototheca moriformis FATA1 allele 1 5′ homology donor regionSEQ ID NO: 150GGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGPrototheca moriformis FATA1 allele 1 3′ homology donor regionSEQ ID NO: 151GACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCTGGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCAATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCCCCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTATTCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCGGAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAATGGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTPrototheca moriformis FATA1 allele 2 5′ homology donor regionSEQ ID NO: 152AATGGAGTCGCTGCTCCACTAATCGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCCGTACTGGCGTCTCTTCCGCTTCTCTGTGCTCCTCTACGCGCTCCGGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCTGTCAGGGCCGCACCCGCAGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGCGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCACAGGACGCGACGTCTTGGTGGCGGTGGCCAGGGACACTGCCCATTGCACAGGCATAGGAATGCGTTCCTTCTCATTTCTCAGTTTTCTGAGCCCCTCCCTCTTCACTCTTTCTCCTCCTCCTCCCCTCTCACGCAGCATTCGTGGPrototheca moriformis FATA1 allele 2 3′ homology donor regionSEQ ID NO: 153CACTAGTATCGATTTCGAACAGAGGAGAGGGTGGCTGGTAGTTGCGGGATGGCTGGTCGCCCGTCGATCCTGCTGCTGCTATTGTCTCCTCCTGCACAAGCCCACCCACGACTCCGAAGAAGAAGAAGAAAACGCGCACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACGGCGATGCCCCCCTCAATCAGCCTCCTCCTCCCTGCCGCTCCAATCTTCCCTGCTTGCATGCGCCCGCGAGAGGCTGTCTGCGCGCCCCGTCAGTCACTCCCCGTGCAGACGCCTCGTGCTCGGTGCTCCTGTATCCTTTACCGCTCCTTTCATTCTGCGAGGCCCCCTGTTGAATGTATTCGTTGCCTGTGTGGCCAAGCGCGCTGCTGGGCGCGCCGCCGTCGGGCGGTGCTCGGCGACTCTGGCGGAAGCCGGTTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTTGGAAAAGAGGGGGGTTTACTGAATGGAGGAGGAGCAGGATAATTGGTAGTATCTGAGTTGTTG SAD2 hairpin C SEQ ID NO: 154actagtGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACTGTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGCATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCGACCAAGTACAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCAGTGTGTTTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccTGCTCGGCCGCAAGGCGCGCGGTGTTGCCGTGGCTGTACTTGGTCGCGCGCTCCTGGAAGGAGGTGTAGACGAAGCCCAAGTAAGGGTTGTTGTCCGTCTGCGGGTTCATGCCGCTCTTGATCAGGTTGTTGATGGTCACCTCCACGGCCCGCATGTTGACGCGCCCCGTCAGCCAACAGTACTTGTTCAGCAGGTCGCCGTGCCGGTTCTCCTCGGCCACCCACTGCCGCGTCCAGCGCaagctt SEQ ID NO: 155MSIQFALRAAYIKGTCQRLSGRGAALGLSRDWTPGWTLPRCWPASAAATAPPRARHQERAIHLTSGRRRHSALASDADERALPSNAPGLVMASQANYFRVRLLPEQEEGELESWSPNVRHTTLLCKPRAMLSKLQMRVMVGDRVIVTAIDPVNMTVHAPPFDPLPATRELVAGEAADMDITVVLNKADLVPEEESAALAQEVASWGPVVLTSTLTGRGLQELERQLGSTTAVLAGPSGAGKSSIINALARAARERPSDASVSNVPEEQVVGEDGRALANPPPFTLADIRNAIPKDCFRKSAAKSLAYLGDLSITGMAVLAYKINSPWLWPLYWFAQGTMFWALFVVGHDCGHQSFSTSKRLNDALAWLGALAAGTWTWALGVLPMLNLYLAPYVWLLVTYLHHHGPSDPREEMPWYRGREWSYMRGGLTTIDRDYGLENKVHHDIGTHVVHH SEQ ID NO: 156MFWALFVVGHDCGHQSFSTSKRLNDAVGLEVHSIIGVPYHGWRISHRTHHNNHGHVENDESWYPPTESGLKAMTDMGRQGRFHFPSMLFVYPFYLFWRSPGKTGSHFSPATDLFALWEAPLIRTSNACQLAWLGALAAGTWALGVLPMLNLYLAPYVISVAWLDLVTYLHHHGPSDPREEMPWYRGREWSYMRGGLTTIDRDYGLENKVHHDIGTHVVHHLFPQIPHYNLCRATKAAKKVLGPYYREPERCPLGLLPVHLLAPLLRSLGQDHFVDDAGSVLFYRRAEGINPWIQKLLPWLGGARRGADAQRDAAQ Camelina sativa omega-3 FAD7-2 SEQ ID NO: 157MANLVLSECGIRPLPRIYTTPRSNFVSNNNKPIFKFRPFTSYKTSSSPLACSRDGFGKNWSLNVSVPLTTTTPIVDESPLKEEEEEKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKSMSYVLRDVAIVFALAAGASYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNNPRLNNVVGHLLHSSILVPYHGWRISHRTHHQNHGHVENDESWHPMSEKIYQSLDKPTRFERFTLPLVMLAYPFYLWARSPGKKGSHYHPESDLFLPKEKTDVLTSTACWTAMAALLICLNEVVGPVQMLKLYGIPYWINVMWLDFVTYLHHHGHEDKLPWYRGKEWSYLRGGLTTLDRDYGVINNIHHDIGTHVIHHLFPQIPHYHLVEATEAVKPVLGKYYREPDKSGPLPLHLLGILAKSIKEDHYVSDEGDVVYYKADPNMYGEIKVGADPrototheca moriformis delta 12 desaturase allele 2 SEQ ID NO: 158MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGIMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVEHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVEVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMENVASRPYPRFANHFDPWSPIFSKRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYMIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDSILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHKCamelina sativa omega-3 FAD7-1 SEQ ID NO: 159MANLVLSECGIRPLPRIYTTPRSNFVSNNNKPIFKFRPLTSYKTSSPLFCSRDGFGRNWSLNVSVPLATTTPIVDESPLEEEEEEEKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKSMSYVLRDVAIVFALAAGAAYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNNPRLNNVVGHLLHSSILVPYHGWRISHRTHHQNHGHVENDESWHPMSEKIYQSLDKPTRFERFTLPLVMLAYPFYLWARSPGKKGSHYHPESDLFLPKEKTDVLTSTACWTAMAALLICLNEVVGPVQMLKLYGIPYWINVMWLDFVTYLHHHGHEDKLPWYRGKEWSYLRGGLTTLDRDYGVINNIHHDIGTHVIHHLFPQIPHYHLVEATEAVKPVLGKYYREPDKSGPLPLHLLGILAKSIKEDHYVSDEGDVVYYKADPNMYGEIKVGADPmFATA-hpB SEQ ID NO: 160actagtCATTCGGGGCAACGAGGTGGGCCCCTCGCAGCGGCTGACGATCACGGCGGTGGCCAACATCCTGCAGGAGGCGGCGGGCAACCACGCGGTGGCCATGTGGGGCCGGAGCGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccGCTCCGGCCCCACATGGCCACCGCGTGGTTGCCCGCCGCCTCCTGCAGGATGTTGGCCACCGCCGTGATCGTCAGCCGCTGCGAGGGGCCCACCTCGTTGCCCCGAATGaagctt PmFATA-hpCSEQ ID NO: 161actagtGGAGGGTTTCGCGACGGACCCGGAGCTGCAGGAGGCGGGTCTCATCTTTGTGATGACGCGCATGCAGATCCAGATGTACCGCTACCCGCGCTGGGGCGACCTGATGCAGGTGGAGACCTGGTTCCAGAGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccTCTGGAACCAGGTCTCCACCTGCATCAGGTCGCCCCAGCGCGGGTAGCGGTACATCTGGATCTGCATGCGCGTCATCACAAAGATGAGACCCGCCTCCTGCAGCTCCGGGTCCGTCGCGAAACCCTCCaagctt PmFATA-hpD SEQ ID NO: 162actagtCGGCGGGCAAGCTGGGCGCGCAGCGCGAGTGGGTGCTGCGCGACAAGCTGACCGGCGAGGCGCTGGGCGCGGCCACCTCGAGCTGGGTCATGATCAACATCCGCACGCGCCGGCCGTGCCGCATGCCGGGTGTGTTTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccCCGGCATGCGGCACGGCCGGCGCGTGCGGATGTTGATCATGACCCAGCTCGAGGTGGCCGCGCCCAGCGCCTCGCCGGTCAGCTTGTCGCGCAGCACCCACTCGCGCTGCGCGCCCAGCTTGCCCGCCGaagctt PmFATA-hpE SEQ ID NO: 163actagtGTCCGCGTCAAGTCGGCCTTCTTCGCGCGCGAGCCGCCGCGCCTGGCGCTGCCGCCCGCGGTCACGCGTGCCAAGCTGCCCAACATCGCGACGCCGGCGCCGCTGCGCGGGCACCGCCAGGTCGCGCGCCGCACCGACATGGACATGAACGGGCACGTGAACAACGTGGCCTACCTGGCCTGGTGCCTGGAGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccTCCAGGCACCAGGCCAGGTAGGCCACGTTGTTCACGTGCCCGTTCATGTCCATGTCGGTGCGGCGCGCGACCTGGCGGTGCCCGCGCAGCGGCGCCGGCGTCGCGATGTTGGGCAGCTTGGCACGCGTGACCGCGGGCGGCAGCGCCAGGCGCGGCGGCTCGCGCGCGAAGAAGGCCGACTTGACGCGGACaagcttPmFATA-hpF SEQ ID NO: 164actagtCCGTGCCCGAGCACGTCTTCAGCGACTACCACCTCTACCAGATGGAGATCGACTTCAAGGCCGAGTGCCACGCGGGCGACGTCATCTCCTCCCAGGCCGAGCAGATCCCGCCCCAGGAGGCGCTCACGCACAACGGCGCCGGCCGCAACCCCTCCTGCTTCGTCCATAGCATTCTGCGCGCCGAGACCGAGCGTGTGTTTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccGCTCGGTCTCGGCGCGCAGAATGCTATGGACGAAGCAGGAGGGGTTGCGGCCGGCGCCGTTGTGCGTGAGCGCCTCCTGGGGCGGGATCTGCTCGGCCTGGGAGGAGATGACGTCGCCCGCGTGGCACTCGGCCTTGAAGTCGATCTCCATCTGGTAGAGGTGGTAGTCGCTGAAGACGTGCTCGGGCACGGaagcttPmFATA-hpG SEQ ID NO: 165actagtTCGTCCGCGCGCGAACCACATGGTCGGCCCCCATCGACGCGCCCGCCGCCAAGCCGCCCAAGGCGAGCCACTGAGGACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccGAGGATATAAGCAGCAGGATCGAATCCCGCGACCAGCGTTTCCCCATCCAGCCAACCACCCTGTCCTCAGTGGCTCGCCTTGGGCGGCTTGGCGGCGGGCGCGTCGATGGGGGCCGACCATGTGGTTCGCGCGCGGACGAaagcttPrototheca moriformis (UTEX 1435) glyoxysomal fatty acid beta-oxidationmultifunctional protein SEQ ID NO: 166ATGTCGAAGGAGCCGCAGTCTTTCTACAAGGTGGAGGATGGAGTGGCCATCATCACCATCTCCTACCCTCCCATGAACGCCCTTCACCCCACGCTGATCAAGGACATGTTCAACAACGTCCGTCGCGCCCAGGAGGATGCCAGCGCCAAGGCCATCGTCATCACCGGTGCCAACGGTCGCTTCTGCGCCGGCTTTGACATCAACCAGTTCCAGAACAAGAGCGCCGGCGCSGGCGGCGGCATCGACATGAGCATCAACAACGCGTTTGTSGAGCTCATCGAGAACGGSCGCAAGCCGACGGTGGCCGCGATCGAGCGGCTGGCCCTGGGCGGCGGGTGCGAGCTGGCGCTGGCCTGCAACGCGCGCGTAGCGGCGCCCGGCACCATCATGGGCCTGCCCGAGCTGACGCTGGGCATTTTGCCCGGCTTTGGCGGCACGCAGCGCCTGCCGCGCGTGGTGGGCCTTGAGAAGGGCCTGTCCATGATCCTGACCAGCAAGCCCATCAAGGARCCCGAGGCGCTCAAGCTGGGCCTGCTGGACGAGGTGGTGCCCGCGCCGCAGCTGCTGGCGCGCGCCAAGGCCTTTGCGCTGGAGATCGTGMCSGGGCAGCGCCCGCGCGTCAACGCGCTCCAGCGCACGGACAAGCTGCCCGCGTACGCGGACGCGCTGCAGATCATCCAGTTTGCGCGCGAGCAGACGGCCAAGCGCGCGCCCAACCTGCAGCACCCGCTCTTCACGCTGGACGCGATCCAGGCCGGCATCGAGCACGGCGGGCTCAAGGGCCTGCGCGCCGAGGCCGACGCCTTTGCGCGCTCGGCGGCGCTGCCCACGCACGACTCGCTGGTGCACGTCTTCTTCGCGCAGCGCTCGACCAAGCGCGTGCGCGGAGTGACCGACGTCGGGCTCAAGCCACGTGCGGTCAAGAGGATCGCCGTGCTGGGCGGCGGGCTCATGGGCTCGGGCATCGCCACCGCCTCGGCGCTGGCGGGCGTSGAGGTGCTGCTCAAGGAYATCAARSAYAAYTTCCTSGAGGGCGGCCTGGGCCGCATCCGCGCCAACCTGGCGTCCAAGGTCAAGCGGAAGCAGCTCTCGCAGCAGGCGGCGGACGCCGCGATGGCCCGCGTCAAGGGCGTGCTCACCTACGACGACTTCAAGACSGTCGACATGGTGATCGAGGCGGCCATCGAGAAGGTGGACCTGAAGCAGGACATCTTCGTGGACCTGGTCAGGGCCACGCGCCCCGACTGCATCCTGGCCACCAACACGTCGACCATCAACATCGAGACGGTGGGCGCCAAGATCCCGGGCGACCTGGGCCGCGTGATCGGCGCGCACTTTTTCTCGCCRGCGCACATCATGCCCCTGCTGGAGATCGTGCGCTCGGACAAGACCAGCGCGCAGGTCATCCTGGACACGCTCGAGTACGGCAGCAAGATCAAGAAGACGCCCGTCGTGGTGGGCAACTGCACGGGCTTTGCGGTCAACCGCGTCTTCTTCCCCTACTCCATGGCCGCGACCATTCTCTTGGACTCGGGGCTCGACCCCTACGCCATCGACGCCGCCATCACGGCCTTTGGCATGCCCATGGGGCCCTTCCGCCTGGGCGACCTGGTCGGCCTGGACGTCTCGCTCTTCGTGGGCGCCTCCTACCTGCAGGACTACGGCGACCGCGTCTACCGCGGCGCCATGGTCCCGCTGCTCAACGAGGCCGGCCGRCTGGGCGAGAAGACCGGCGCCGGCTGGTACAAGTTTGACGACCGCCGCCGCGCGAGCCCGGACCCGGCGCTGGCGCCGCTCCTCTCGCCCAAGGACATCGCCGAGTTCATCTTCTTCCCGGTCGTCAACGAGGCCTGCCGCGTCGTGGCCGAGGGCATCGTGGACAAGCCCTCCGACCTGGACATCGCCACCGTCATGTCCATGGGCTTCCCGGCCTACCGCGGCGGCATCCTCTTCTACGGCGACATCGTCGGCGCCAAGTACGTCGTCGACCGTCTCAACGCCTGGGCCAAGCAGTACCCCGCGCAGGCCGGCTTCTTCAAGCCCTGCGACTACCTCCTSCGCGCCGCGCAGACCGGCACCAAGCTCGAGGCCGGCAACAAGCCCGCCTCCAAGATCTGAPrototheca moriformis (UTEX 1435) glyoxysomal fatty acid beta-oxidationmultifunctional protein SEQ ID NO: 167MSKEPQSFYKVEDGVAIITISYPPMNALHPTLIKDMENNVRRAQEDASAKAIVITGANGRFCAGFDINQFQNKSAGXGGGIDMSINNAFXELIENXRKPTVAAIERLALGGGCELALACNARVAAPGTIMGLPELTLGILPGFGGTQRLPRVVGLEKGLSMILTSKPIKXPEALKLGLLDEVVPAPQLLARAKAFALEIVXGQRPRVNALQRTDKLPAYADALQIIQFAREQTAKRAPNLQHPLFTLDAIQAGIEHGGLKGLRAEADAFARSAALPTHDSLVHVFFAQRSTKRVRGVTDVGLKPRAVKRIAVLGGGLMGSGIATASALAGXEVLLKXIXXXFXEGGLGRIRANLASKVKRKQLSQQAADAAMARVKGVLTYDDFKXVDMVIEAAIEKVDLKQDIFVDLVRATRPDCILATNTSTINIETVGAKIPGDLGRVIGAHFFSXAHIMPLLEIVRSDKTSAQVILDTLEYGSKIKKTPVVVGNCTGFAVNRVFFPYSMAATILLDSGLDPYAIDAAITAFGMPMGPFRLGDLVGLDVSLFVGASYLQDYGDRVYRGAMVPLLNEAGXLGEKTGAGWYKFDDRRRASPDPALAPLLSPKDIAEFIFFPVVNEACRVVAEGIVDKPSDLDIATVMSMGFPAYRGGILFYGDIVGAKYVVDRLNAWAKQYPAQAGFFKPCDYLXRAAQTGTKLEAGNKPASKIPrototheca moriformis (UTEX 1435) Triacylglycerol Lipase (TAGL)SEQ ID NO: 168ATGGACAGGGCGTGGATGCTGGACCGCGAGCTGGCGGCCAACCGCAGCCGGGGCCTTGACTGGGGCGTGGCGCGGGCGCTGTCGGCCTACGTCTCGGCCTCGTACTGCAACAGCACGAGCCTGGCCGCGTGGAACTGCACGCGCTGCTCGCCGCGCGGCATCGACGGCAACGCCACGTGGATRGACCCCAACTTTGCGCTGGAGGCGCTGGCCTGGGACGAGGCCTGGGACCTGCTGGGCTACGTGGGCTGGAGCGAGGAGATGGGCGCGACCGTGATCGCCTTCCGCGGCACCGACTCGCACAGCTACCACAACTGGGTGGCCAACATGCGCACCTGGCGCACCGACCTCAACCTGACCATGCACGGCGCGCCGGCCAACGCGCTCGTGCACGGCGGCTTCTGGTTCTCCTGGAACGCCAGCTCCCTGGCCACCTCCGTGACCGCGGCCGTGCTGCGCCTGCAGCGGCGCCACGGCGCGCACCCGGTCTACGTGTCGGGGCACTCGCTGGGCGGCGCGCTGGCCACCATCTGCGCGCTGGAGCTGCGCACCATGCYCCYCKTGCGCGACGTGCACCTGGTCACCTTTGGCAGCCCGCGCGTGGGCAACGCCGTCTTCGCCGGCTGGTTCGAGCGGCGCATCACCTCGCACTGGCGCTTCACGCACAACCGCGARATCGTGCCCAGCGTGCCGCCGCCCTACATGGGCTTCTGGCACCTGGCGCGCGAGGTCTGGGTGCTGGACAACGCGCGCCTCAACCCGCTCGTCGGCGTCTGCGACGGGACGGGGGAGGACATGCAGTGCCACAACTCCATGTGCCACCTGGGCCTCTGCTCCTCCATCGCAGACCACCTCCTCTACATCTCKGAGATGTACACCCCGAGGCCCATGGGCTGCTGAPrototheca moriformis (UTEX 1435) Triacylglycerol Lipase (TAGL)SEQ ID NO: 169MDRAWMLDRELAANRSRGLDWGVARALSAYVSASYCNSTSLAAWNCTRCSPRGIDGNATWXDPNFALEALAWDEAWDLLGYVGWSEEMGATVIAFRGTDSHSYHNWVANMRTWRTDLNLTMHGAPANALVHGGEWFSWNASSLATSVTAAVLRLQRRHGAHPVYVSGHSLGGALATICALELRTMXXXRDVHLVTEGSPRVGNAVFAGWEERRITSHWRFTHNRXIVPSVPPPYMGEWHLAREVWVLDNARLNPLVGVCDGTGEDMQCHNSMCHLGLCSSIADHLLYIXEMYTPRPMGCPrototheca moriformis (UTEX 1435) Fumarate hydratase SEQ ID NO: 170ATGAGTACCCGCACGGAGCGCGACACGTTTGGGCCCCTGGAGGTTCCCTCCGACAGGTACTGGGGTGCCCAGACGCAGCGTTCCCTGCAAAACTTCAAGATCGGCGGCGCCCCCGAGCGCATGCCGGAGCCGGTGGTGCGTGCCTTTGGCGTGCTCAAGGGCGCGGCGGCCAAGGTGAACATGGACCTGGGCGTGCTGGACAAGACCAAGGGCGAGGCGATCGTGGCGGCGGCCAAGGAGGTGGCGGAGGGCAAGCTGACGGACCACTTCCCCCTGGTGGTGTGGCAGACGGGGTCTGGCACGCAGAGCAACATGAACGCCAACGAGGTGATTGCCAACCGCGCGATCGAGATGCTGGGCGGCGAGCTGGGCGACAAGAGCGTGGTGCACCCCAACGACCACTGCAACAAGGGCCAGAGCTCCAACGACACCTTCCCCACGGTGATGCACATCGCGGGCGTGACGGAGATCAGCCGCCGCCTGGTGCCCGGGTTGCGCCACCTGGCGGAGGCGCTGCGCGCCAAGGAGAAGGAGTTTGCCGGCATCATCAAGATCGGGCGCACGCACACGCAGGACGCGACGCCGCTGACGCTGGGCCAGGAGTTCAGCGGCTACGCGACGCAGGTCGAGTACGGCATCGAGCGCGTGCAGGCCGCGCTGCCGCGGCTGAGCATGCTGGCGCAGGGCGGCACCGCGGTGGGCACGGGGCTGAACGCCAAGGTCGGCTTTGCGGAGAAGGTGGCCGAGGCGGTGGCCGCCGACACGGGCCTGCCCTTCACCACCGCGCCCAACAAGTTTGAGGCGCTGGCCGCGCACGACGCGGTGGTGGAGGCCAGCGGCGCGCTCAACACCGTGGCTGTGAGCCTGATGAAGGTCGCCAACGACGTGCGCTTCCTGGGCAGCGGCCCGCGCTGCGGCCTGGGCGAGCTGAGCCTGCCCGAGAACGAGCCGGGCTCCTCCATCATGCCGGGCAAGGTCAACCCCACGCAGTGTGAGGCGCTGACCATGGTCTGCGCGCAGGTGATGGGCAACCACGTGGCCGTCAGCGTGGGCGGCTCCAACGGCCACTTTGAGCTCAACGTGTTCAAGCCCATGATGATCCGCAACCTGCTGCACTCGGCGCGCCTGCTCGCCGACGCCGCCACCTCCTTCACCGACAACTGCGTCGTGGGCATCAGGGCCAACGAGGGCCGCATCGCGCAGCTCCTGCACGAGTCCCTCATGCTCGTCACCGCGCTCAACAACCACATCGGCTACGACAAGGCCGCCGCCATCGCCAAGAAGGCGCACAAGGACGGCAGCACGCTCAAGCAGGCCGCGCTCGAGCTCGGCTACTGCACCGAGCAGGAGTTTGCCGACTGGGTCAAGCCCGAGGACATGCTCGCGCCCACGCCCTAGPrototheca moriformis (UTEX 1435) Fumarate hydratase SEQ ID NO: 171MSTRTERDTFGPLEVPSDRYWGAQTQRSLQNFKIGGAPERMPEPVVRAFGVLKGAAAKVNMDLGVLDKTKGEAIVAAAKEVAEGKLTDHFPLVVWQTGSGTQSNMNANEVIANRAIEMLGGELGDKSVVHPNDHCNKGQSSNDTEPTVMHIAGVTEISRRLVPGLRHLAEALRAKEKEFAGIIKIGRTHTQDATPLTLGQEFSGYATQVEYGIERVQAALPRLSMLAQGGTAVGTGLNAKVGFAEKVAEAVAADTGLPFTTAPNKFEALAAHDAVVEASGALNTVAVSLMKVANDVRFLGSGPRCGLGELSLPENEPGSSIMPGKVNPTQCEALTMVCAQVMGNHVAVSVGGSNGHFELNVEKPMMIRNLLHSARLLADAATSFTDNCVVGIRANEGRIAQLLHESLMLVTALNNHIGYDKAAAIAKKAHKDGSTLKQAALELGYCTEQEFADWVKPEDMLAPTPPrototheca moriformis (UTEX 1435) succinate semialdehyde dehydrogenaseSEQ ID NO: 172ATGTCGACCRTGTCCGAATCCAAGGAGATCCARGTCCGGGACGATGTCCTGAAGCGGCTCAARGACCCATCCCTCRTGCACACCGAGTCGTACATCGGGGGCAGCTGGGTGAACGCTGCCGATGACGACAGGGTGGAGGTGCACGACCCGGCCAGTGGCGAGGTGCTCGCCCGCGTGACTCATGCCAAGGCTGTGGAGACCAAGGCGGCCATCGCSGAGGCGTCCTCTGTGTTTGGCATGTGGTCGGCRAAGTCMGGCCWGGAGCGGTCCAACATCCTRCGCCGCTGGTTTGAGCTGCTGCGTGAGAATCAGGACGACATRGCGACGCTCATGACCCTGGAGAGCGGCAAGCCCCTGGSCCAGGCCAAGGCCGAGATCGCGAGCGGCCTGGGCTCTGTGGAGTGGTTTGCGGAGGAGGCCAAGCGCGTGGACGGCGACATTCTGTGCAGCCCCTTCCCCAACAAGCGCTACCTGGTCATGCGGCAGCCCATCGGCGTGGTGGGCGCCATCACGCCCTGGAACTTTCCCTTTTCCATGATCACGCGCAAGATCTCGCCCGCCCTGGCGGCCGGCTGCACGGTGGTGCTGAAGCCGAGCGAGCTGACGCCGCTGACGGCGCTGGCGATGGCCGAGCTGGGGGAGCGGGCCGGCATCCCGCTGGGCGTGCTCAACGTCGTCGTGGGCGACGCCAAGTCCATCGGCGACGAGCTCATCAAGAGCGACGAGGTGCGCAAGGTCGCCTTCACCGGCTCCACCCGCATCGGCAAGATCCTCATGGCCGGCGCGGCCAACACGGTCAAGAAGGTCTCCATGGAGCTGGGCGGGAACGCGCCCTACATCGTCTTCCCAGACGCCGACCTCAAGGCGGCGGCCTCCCAGGCGGCGGCGTCCTCCCACCGCAACGCCGGGCAGACGTGCATCTGCACCAACCGGGTGCTTGTCCACGAAAGCGTGCACGACGAGTTTGTCAAGGAGCTGGTGGCAGCGGTGCAGGAATTCAGGCTGGGTCACGGCACCGACCCCAGCACCACGCACGGCCCGCTCATCCAGCCGGGGGCCGTCGACAAGGTGGAGGAGAAGGTGCAGGACGCGGTGGCCAAGGGCGCGACGGTGCAGACCGGGGGCAAGCGCCCCACGTTCAGCGAGAACAGCCCCCTCAACAACGGCTTCTTCTTCGAGCCCACCGTCATCTCCAATGCGACCATCGACATGAAGGTGTTCAGGGAGGAGATCTTCGGGCCCGTGACCCCCGTGTTCAAGTTTTCCTCGGACGAGGAGGCCGTCAAGCTGGCGAACAACACCGAGTACGGGCTGGCTGCCTACCTCTTCACCAAGGACCTCGCGCGCGCCTGGAAGGTGTCCGAGGCGCTCGAGTTCGGCATGGTCGGCGTGAACGACGTGCTCATCACCAGCTACGTCTCGCCCTTTGGCGGCGTCAAGCAGTCGGGCCTSGGCCGCGAGGAGTCCAAGTACGGCATCGARGAGTACCTGCAGATGAAGTCGGTGTGCATGAACCTGGGGTACTGAPrototheca moriformis (UTEX 1435) succinate semialdehyde dehydrogenaseSEQ ID NO: 173MSTXSESKEIXVRDDVLKRLXDPSLXHTESYIGGSWVNAADDDRVEVHDPASGEVLARVTHAKAVETKAAIXEASSVFGMWSXKXGXERSNIXRRWFELLRENQDDXATLMTLESGKPLXQAKAEIASGLGSVEWFAEEAKRVDGDILCSPFPNKRYLVMRQPIGVVGAITPWNFPFSMITRKISPALAAGCTVVLKPSELTPLTALAMAELGERAGIPLGVLNVVVGDAKSIGDELIKSDEVRKVAFTGSTRIGKILMAGAANTVKKVSMELGGNAPYIVFPDADLKAAASQAAASSHRNAGQTCICTNRVLVHESVHDEFVKELVAAVQEFRLGHGTDPSTTHGPLIQPGAVDKVEEKVQDAVAKGATVQTGGKRPTFSENSPLNNGFFFEPTVISNATIDMKVFREEIFGPVTPVFKFSSDEEAVKLANNTEYGLAAYLFTKDLARAWKVSEALEFGMVGVNDVLITSYVSPFGGVKQSGXGREESKYGIXEYLQMKSVCMNLGYPrototheca moriformis (UTEX 1435) Malonyl coenzyme A: acyl carrier proteintransacylase SEQ ID NO: 174ATGGCCGCTACGCTCAGCATGAAGTCCGTGGCGCTGCAGCGTCCCACCAGGTGTGGGCCTGCACCCCGCACAGTGGTCCGGGCGCCGGCCATGCGCGTCCGGGCTGCAGCCACCGCCGCCCCGGAGGCCCCCGCCGCGGACCACGCGTTTGACGACTACCAGCCCCGCACGGCCATCCTCTTCCCTGGGCAGGGCGCGCAGAGCGTGGGCATGGCGGGCGAGCTRGTCAAGTCGGTGCCCAAGGCGGCSCACATGTTTGACGAGGCCTCYGCCATCCTKGGCTACGACCTGCTCAAGACSTGCGTGGAGGGCCCCAAGGAGCGCCTGGACAGCACGGTGGTGAGCCAGCCGGCCATCTACGTGACGAGCCTGGCGGCCGTCGAGAAGCTGCGCGCCGACGAGGGCGACGAGGCGGTGGCGGCCGTCGACGTGGCGGCGGGCCTGTCCCTGGGCGAGTACACGGCCCTGGCCTTTGCCGGCGCCTTCAGCTTCGAGGACGGGCTGCGACTGGTCGCGCTGCGCGGCGCCAGCATGCAGCGCGCGGCCGACGCGGCCGCCAGCGGCATGGTCTCCGTCATCGGCCTCTCGGCCGCCGCGACCGAGGCGCTGTGCGAGGCGGCCAACGCCGAGGTCCCGCCCGAGCAGGCCGTGCGCGTGGCCAACTACCTCTGCAACGGCAACTACGCCGTGAGCGGCGGCCTYGAGGGCTGCGCGGCCGTCGAGCGCCTRGCCAAGGCGCACAARGCGCGCATGACCGTGCGCCTGGCCGTGGCCGGCGCCTTCCACACGGCCTACATGCAGCCGGCCGTGGAGGCGCTGACCGCGGCCCTGGCCAGCACGGACATCGCCGCRCCGCGCATCCCCGTCGTCTCCAACGTCGACGCYGCGCCGCACGCCGACCCGGACACCATCCGCGCCCTGCTGGCCCGCCAGGTCACCGCGCCCGTGCAGTGGGAGACCACGCTCAACACCCTGCTCTCCCGCGGCCTCCAGCGCTCCTACGAGGTCGGCCCGGGCAAGGTCATCGCCGGCATCATCAAGCGCGTCAACAAGAAGCACCCGGTGACCAACATCACTGCCTGAPrototheca moriformis (UTEX 1435) malonyl coenzyme A: acyl carrier proteintransacylase SEQ ID NO: 175MAATLSMKSVALQRPTRCGPAPRTVVRAPAMRVRAAATAAPEAPAADHAFDDYQPRTAILFPGQGAQSVGMAGEXVKSVPKAXHMFDEAXAIXGYDLLKXCVEGPKERLDSTVVSQPAIYVTSLAAVEKLRADEGDEAVAAVDVAAGLSLGEYTALAFAGAFSFEDGLRLVALRGASMQRAADAAASGMVSVIGLSAAATEALCEAANAEVPPEQAVRVANYLCNGNYAVSGGXEGCAAVERXAKAHXARMTVRLAVAGAFHTAYMQPAVEALTAALASTDIAXPRIPVVSNVDXAPHADPDTIRALLARQVTAPVQWETTLNTLLSRGLQRSYEVGPGKVIAGIIKRVNKKHPVTNITAPrototheca moriformis (UTEX 1435) phosphatidate cytidylyltransferaseSEQ ID NO: 176ATGCAGTTGCAGCGAGCGCTCACGGTGCGCCTGCGGAGGAGGGGCTGGCGTCGCAACTCGTTCAACAATGTGGACGCCGTCTCGCCCAAGCTTGTGCGCAAGWCATCCAATGCCGGGCAGCAGACACAGTCGGAGGACAGGTTTAGGTCCTTCAAGGTCAGGACCCTGTCCACAATCGTCCTCATCGCCACCTTCATCGGCATCATCGCCACGGGGCACGTGCCCCTCATGCTCATGATCCTGGGCATCCAGTTTCTGATGGTCMGGGAGCTSTTTGCGCTGGCGAGGGTSGCGCCCCAGGAGCGCAAGGTCCCCGGCTTCCGGGCGCAGCAGTGGTACTTTTTCTTCGTGGCCGCCTTTTACCTGTACATCCGCTTCATCAAGACCAACCTCACGGTGGAGCTCTCCTCCTCGGCGCAAACGGCGGCCATGTTTGGCTGGATCATCCGCCACCACACGCTCCTCTCCTTCGCCCTCTACACGGCCGGGTTCGTGTCGTTCGTGCTGCGCCTGAARAAGGGACTCTACAGYTACCAGTTCCAGCAGTACGCCTGGACGCACATGATCCTCATGGTCATCTTCCTGCCTTCGTCCTTCTTTGTGTACAACCTCTTCGAGGGGCTGATCTGGTTCCTGCTCCCCGCGGCCTTGGTCGTGGTCAACGACATYGCGGCCTACCTGGCAGGCTTTTTCTTTGGGAGGACCCCKCTGATCAAGCTGTCTCCCAAGAAGACGTGGGAGGGCTTYATCGGCGGCCTGGCGGGCACGGTGGTGGTGGGCTGGTATCTGGCCAAGTTGCTGTCTCAATTCAACTGGTTCATCTGTCCTCGGCGGGACCTGTCCATCATCCAGCCTCTGCACTGTGACAGCAAGCTGGACATCTACCAGCCAGAGACATTCTACCTGACAGATGTCTTGACGCTCTTGCCGGCCGACCTGGCCACCGCGGTGGTCACGGGGCTCACCCGCCTCTCGCCCGGCTGGCAGGAAGCTGCGCGGCAGGTGTCCCTGACCTGCCTGCCCATGCAGCTGCACGCGGTCGTGCTGGCGACGTTTGCGAGCCTGATCGCACCCTTTGGCGGCTTCTTCGCCTCGGGCTTCAAGCGCGGCTTCAAGATCAAGGACTTTGGCGACAGCATCCCCGGCCACGGCGGCATGACCGACCGCATGGACTGCCAGGTGGTCATGTCGGTCTTCTCCTWCATCTACCTCACGTCCTACGTGGCGCCCGCGGGCCCCACCGTCGGCAGCGTCCTGGCGGCCGCCGTGAAGCTGGCGCCGCTGGARCAGCTGGACCTGTTTGAGCGCATGGCCAACGTGCTGGTGGGCTCCAAGGTCCTGTCCCCGGCCATCGGCGAGACCATCGCCGCCACCGCGCGCCGCAAGCTCCAGACGGGCGCCCTTTAGPrototheca moriformis (UTEX 1435) phosphatidate cytidylyltransferaseSEQ ID NO: 177MQLQRALTVRLRRRGWRRNSFNNVDAVSPKLVRKXSNAGQQTQSEDRFRSFKVRTLSTIVLIATFIGIIATGHVPLMLMILGIQFLMVXEXFALARXAPQERKVPGFRAQQWYFFFVAAFYLYIRFIKTNLTVELSSSAQTAAMFGWIIRHHTLLSFALYTAGFVSFVLRLXKGLYXYQFQQYAWTHMILMVIFLPSSFFVYNLFEGLIWFLLPAALVVVNDXAAYLAGFFFGRTXLIKLSPKKTWEGXIGGLAGTVVVGWYLAKLLSQFNWFICPRRDLSIIQPLHCDSKLDIYQPETFYLTDVLTLLPADLATAVVTGLTRLSPGWQEAARQVSLTCLPMQLHAVVLATFASLIAPFGGFFASGFKRGFKIKDFGDSIPGHGGMTDRMDCQVVMSVFSXIYLTSYVAPAGPTVGSVLAAAVKLAPLXQLDLFERMANVLVGSKVLSPAIGETIAATARRKLQTGALPrototheca moriformis (UTEX 1435) 1-acyl-sn-glycerol-3-phosphateacyltransferase, putative SEQ ID NO: 178ATGTTGCTGCCCTTTGAGCTCATGTTCGACCGCTACAGGCGGCATATTCTGAACAAGATCAACCAGTTTTGGGCGCGGGTGGCCGCGTGGCCGTTTTACGGCGTCATCGTGGAYGGCCGCGAGAACCTGCCRCCTCCCGGCGAAGCCGTGATCTATGTCGCCAACCACCAGAGCTACCTGGACATCACGGCGCTGCTGCACATGGGCGCMGACTTCAAGTACGTCTCCAAGGCCGCCCTTTTCATGGTGCCTTTCCTGGGCTGGGCCATGTTCCTCACAGGGCACGTCCGGCTCGTRCGAGACGACCGCGAGTCGCAGCAAAAATGCTTGAAACAGTGCGACACCCTCCTCCGCAACGGCACCTCCGTCGCCTTTTTCCCCGAGGGCACGCGGAGCCAGGATGGSAAGATGCATGCCTTCAAAAAGGGCGCCTTTATGGTGGCGGCCAGGGCGCGGGTGAACGTCGTGCCCGTCACGATCGAGGGCTCCGGCGACCTGATGCCCAGCCGGAACGAGTACCTGATGACCTACGGCGACATTCGGGTGCGCATCCACCCGCCCATCGCCTCGCGCGGCCGGTCGCTGGAGTCGCTGCGCCTGGCGGCGCAGGAGGCGGTGGCGTCCGSGCTGCCCGAGCGCCTGATCGGGGACGACATCCGGCCGCGCKAGCTGGAGGCGCCCACGCCGGCGCCGACAGAGGCGACAGAGGCGAGAGAGGCGCCGCTGCACGAACAGCGCTGAPrototheca moriformis 1-acyl-sn-glycerol-3-phosphate acyltransferase,putative SEQ ID NO: 179MLLPFELMFDRYRRHILNKINQFWARVAAWPFYGVIVXGRENLXPPGEAVIYVANHQSYLDITALLHMGXDFKYVSKAALFMVPFLGWAMFLTGHVRLXRDDRESQQKCLKQCDTLLRNGTSVAFFPEGTRSQDXKMHAFKKGAFMVAARARVNVVPVTIEGSGDLMPSRNEYLMTYGDIRVRIHPPIASRGRSLESLRLAAQEAVASXLPERLIGDDIRPRXLEAPTPAPTEATEAREAPLHEQRPrototheca moriformis (UTEX 1435) glycerophosphodiester phosphodiesteraseSEQ ID NO: 180ATGTCCAAGGCTTCTTCCACCATCGTCTCCGCTCCTGCGGGCCCGCAGGAGTCGCCCGCTCGCTACAGTACCGAGTCGACCGAGATCCGGAGCTCYCCCCTGTTTGGACGCCTTTTGGACGTCAAGAGCAACGTTGTCGCCCTTGGAGGGCACAGAGGYCTGGGCGCCAACTACTGGGACAAGCCGGCGCAGGGCTCGCTCCGCCGCTTCAGCGTGCGAGAGAACACCATCCCCTCCTTCCTCAAGTCGGTTGCGGCCGGCGCCAGCTTCATCGAGTTTGACGTGCAGGTCACGCGGGACGGCGTGCCTGTGATCTGGCACGACAACTATGTGGTAACGGGCACGCCCGATTGCTTTGTGAACCGGCTGATCAGCGACCTGAGCTACGACGAGTTTGTGAGCCTGGTGCCGGCCAGCGCCGAYCTGGCGCGCCAGAAGGCGGGCCAGGTGGCGGGCCTGCGCGCGGAGGACGTGGAGGGCAGCACGCGCTTGCTGCGCGCGCTGGTGGGCGACGAGCCRGTGGACCCSGGCAACCCGACCGTGGCGGGGTGGGAGGCCGACTCGGAGTCGCGCCTGCCCAGCTTGGCCGAGCTGTTCGCAGCCATCCCCTCGCACGTCGCCTTTGACATCGAGATCAAGGTCGCGACCAGCAGCCGCACGGTGCACACGCCGCTGCGCGAGGTGGAGCGCCTGCTGTCCGCCATCCTGACGGCCGTGGACGCGGCGCAGGAGGCGGCCGAGGCCGCGGGCCGCCCGCGGCGCGAGATCCTGTACAGCTCCTTCGACCCGGACGTCTGCGCGGAGCTGGCGCAGCGCCGCCCCGACGCGGCCGTCATGTTCCTGTCGGGCGGCGGGCAGTATGCGCACTCGGACCCCCGCCGCACCTCCTTTGCGGCCGCCATCGACTGGGCGCGCGGCGCCAACCTGGCCGGCGTGATCTTCCACGCGGGCCGCCTGCGCACCGCGCTCGAGGCGGTCGTGCGCGACGCGCTGGCCGCGGGCCTCCAGGTCATGACCTACGGCCAGGACAACACCGACGCCGGCTACGTGGAGGAGCAGTACGCTCGCGGCGTCCGCGGCGTCATCGTGGACGACGTCAGCTCGGTCGTGGACGCGCTCGTCCAGCGCGGCGTCGCGCGGCGCTCGGTCGCGCTGGCCGCCGGCGAGGGCAAGCAGAACGGCGTGCCGGACTCGGAGAAGGTGAAGGCCTCGCCCGCGCGCCTGCCGGCCCTGGCCGCCGCCTGCTAAPrototheca moriformis (UTEX 1435) glycerophosphodiester phosphodiesteraseSEQ ID NO: 181MSKASSTIVSAPAGPQESPARYSTESTEIRSXPLFGRLLDVKSNVVALGGHRXLGANYWDKPAQGSLRRFSVRENTIPSFLKSVAAGASFIEFDVQVTRDGVPVIWHDNYVVTGTPDCFVNRLISDLSYDEFVSLVPASAXLARQKAGQVAGLRAEDVEGSTRLLRALVGDEXVDXGNPTVAGWEADSESRLPSLAELFAAIPSHVAFDIEIKVATSSRTVHTPLREVERLLSAILTAVDAAQEAAEAAGRPRREILYSSFDPDVCAELAQRRPDAAVMELSGGGQYAHSDPRRTSFAAAIDWARGANLAGVIFHAGRLRTALEAVVRDALAAGLQVMTYGQDNTDAGYVEEQYARGVRGVIVDDVSSVVDALVQRGVARRSVALAAGEGKQNGVPDSEKVKASPARLPALAAACPrototheca moriformis (UTEX 1435) diacylglycerol acyltransferase type 2SEQ ID NO: 182ATGACGGAGGCCGTCTCGGACAGGGATGCTCTSCAACCGGAGCTGGGCTTCGTSCAGAAGCTGGGCATGTACTTTGCGCTGCCAGTTTGGATGATAGGCCTCGTGGTGGGCGCCCTCTGGCTMCCCGTGACCGTGGTGTGCCTCTTCATCTTCCCAAAGCTGGCGACATTGAGTCTGGGGCTGCTGCTGGTGGCGATGCTCACCCCCCTGTCGCTGCCCTGCCCCAAGCCGCTGGCCCGCTTCCTGGCCTACTGCACCACCGCCGCGGCCGAGTACTACCCCGTGCGSTTCATCTACGAGGACAAGGAGGAGATGGAGGCCACCAAGGGCCCCGTCATCATCGGGTACGAGCCCCACAGCGTCATGCCRCAGGCCATCTCCATGTTRGCCGAGTACCCGCACCCCGCCGTGGTCGGCCCGCTGCGCAAGGCGCGCGTGCTGGCKTCCAGCACCGGCTTCTGGACGCCGGGCATGCGGCACCTGTGGTGGTGGCTGGGCACGCGCCCCGTGAGCAAGCCCTCCTTCCTCGCCCAGCTGCGCAAGCAGCGCTCCGTCGCCCTCTGCCCGGGCGGCGTGCAGGAGTGCCTCTACATGGCCCACGGCAAGGAGGTCGTCTACCTCCGCAAACGCTTTGGATTTGTCAAACTGGCCATCCAGACCGGCACCCCGCTGGTCCCGGTCTTCGCCTTTGGCCAGACGGAGACCTACACCTTTGTGCGGCCCTTCATCGACTGGGAGACGCGCCTGCTGCCGCGGTCCAAGTACTTTTCGCTGGTGCGCCGCATGGGCTACGTGCCCATGATCTTCTTCGGCCACCTGGGCACGGCCATGCCCAAGCGCGTGCCCATCCACATCGTCATCGGCCGGCCCATCGAGGTGCCGCAGCAGGACGAGCCCGACCCGGCCACGGTGCAGCAGTACCTCGACAAGTTCATCGACGCCATGCAGGCAATGTTTGAGAAGCACAAGGCCGACGCCGGCTACCCCAACCTGACGCTCGAGATCCACTGAPrototheca moriformis (UTEX 1435) diacylglycerol acyltransferase type 2SEQ ID NO: 183MTEAVSDRDAXQPELGFXQKLGMYFALPVWMIGLVVGALWXPVTVVCLFIFPKLATLSLGLLLVAMLTPLSLPCPKPLARFLAYCTTAAAEYYPVXFIYEDKEEMEATKGPVIIGYEPHSVMXQAISMXAEYPHPAVVGPLRKARVLXSSTGEWTPGMRHLWWWLGTRPVSKPSFLAQLRKQRSVALCPGGVQECLYMAHGKEVVYLRKREGFVKLAIQTGTPLVPVFAFGQTETYTEVRPFIDWETRLLPRSKYFSLVRRMGYVPMIFFGHLGTAMPKRVPIHIVIGRPIEVPQQDEPDPATVQQYLDKFIDAMQAMFEKHKADAGYPNLTLEIHPrototheca moriformis (UTEX 1435) Ketoacyl-CoA Reductase (KCR)SEQ ID NO: 184ATGGACGCTCAAGGATACCTCGTSAAGGCGTCCGAGTCKCCAGCATGGACGTACCTGGTSCTGCTGGCCTCGGCGCTGTTCGCCATCAAGGTCGTGGGATTYGTCCTGACGGTTCTGGGCGGGCTGTACGCCCACTTTCTCCGCAAGGGAAAGAAACTGCGGCGATATGGGGACTGGGCAGTGGTGACTGGCGCGACAGACGGCATCGGCAAGGCCTACGCTGAGGCCCTCGCGAAGCAAAAGCTGCGCCTGGTGCTGATCTCGCGGACAGAGTCGCGTCTTGAGGAGGAGGCCCGGCTGCTGCAGGACAAGTTTGGCGTGGAGGTGAAGATCATCCCCGCCGACCTCAGCTCCTCGGACGAGGCCGTGTTTGCCCGGATCGGCAAGGGCCTGGAGGGCCTGGACATTGGCATCCTGGTGAACAATGCCGGCATGTCCTACCCCCACCCGGAGTACCTGCACCTGGTCGACGACGAGACCCTGGCCAACCTCATCAATCTCAACGTCGCCACCCCGACCAAGCTGTGCAAGATGGTGCTGGGCGGCATGAAGGAGCGCGGCCGCGGCYTGGTGGTCAACGTYGGSAGCGGCGTCGCGAGCGCCATTCCCTCGGGGCCCCTCCTCTCGGCCTACACCGCCAGCAAGGCGTACGTGGACCAGCTGAGCGAGTCGCTGAACGACGAGTACAAGGAGTTTGGCGTGCAGGTGCAGAACCAGGCGCCGCTGTTTGTGGCCACCAAGATGTCCAAGATCCGCAAGCCGCGCATCGACGCGCCCACGCCCGGCACCTGGGCCGCCRCCGCSGTYCGCGCCATGGGRTTCGAGACGCTGTCCTTCCCCTACTGGTTCCACGCGCTGCAGGCGGCCGTCGTGGAGCGCCTGCCGGAGGCCATGATCCGCTACCAGGTCATGCAGATCCACCGCAGCCTGCGCCGCGCGGCCTACAAGAAGAAGGCGCGCGCGTCCGCCGCCGCCCTGGCGGACGAGGGCGTCTCCGCGGAGCCCAAGAAGGACCTGTGAPrototheca moriformis (UTEX 1435) Ketoacyl-CoA Reductase (KCR)SEQ ID NO: 185MDAQGYLXKASEXPAWTYLXLLASALFAIKVVGXVLTVLGGLYAHFLRKGKKLRRYGDWAVVTGATDGIGKAYAEALAKQKLRLVLISRTESRLEEEARLLQDKFGVEVKIIPADLSSSDEAVFARIGKGLEGLDIGILVNNAGMSYPHPEYLHLVDDETLANLINLNVATPTKLCKMVLGGMKERGRGXVVNXXSGVASAIPSGPLLSAYTASKAYVDQLSESLNDEYKEFGVQVQNQAPLEVATKMSKIRKPRIDAPTPGTWAAXXXRAMXFETLSFPYWFHALQAAVVERLPEAMIRYQVMQIHRSLRRAAYKKKARASAAALADEGVSAEPKKDLPrototheca moriformis (UTEX 1435) glycerol-3-phosphate acyltransferaseSEQ ID NO: 186ATGGAGAGGAGGACCTCGGCCAACCCCTCCGTGGGTCTTAGCAGCTTAAAGCACGTTCCGTTGAGCGCGGTGGACCTCCCCAATTTCGAGTCCGTTCCCAAGGGTGCGGCCAGCCCCGATGTCCATCGTCAGATAGAGGAGCTGGTGGACAAGGCACGCGCAGATCGGTCACCGGCATCGTTGTCGCTCCTAGCCGATGTGCTGGACATCAGCGGGCCGCTGCAGGACGCGGCCTCGGCGATGGTGGACGACTCCTTCCTGCGCTGCTTCACCTCCACCATGGACGAGCCCTGGAACTGGAACTTTTACCTGTTCCCGCTCTGGGCGCTCGGCGTCGTCGTRCGCAACRTGATCCTGTTCCCCCTGCGCCTCCTCACCATCGTGCTGGGGACGCTGCTCTTCGTGCTGGCCTTTGCCGTGACGGGCTTCCTCCCCAAGGAGAGGCGGCTCGCGGCCGAGCAGCGGTGCGTCCAGTTCATGGCGCAGGCATTCGTGGCCTCCTGGACAGGCGTGATCCGGTACCACGGCCCCCGCCCCGTGGCGGCGCCCAACCGCGTGTGGGTCGCCAACCACACGTCCATGATCGACTACGCGGTGCTGTGCGCCTACTGCCCGTTTGCGGCCATCATGCAGCTGCACCCSGGCTGGGTGGGCGTCTTRCAGACGCGCTACCTGGCCTCGCTGGGCTGCCTCTGGTTCAACCGCACGCAGGCCAAGGACCGCACGCTCGTGGCCAAGCGCATGCGCGAGCACGTGGCCAGCGCGACCAGCACGCCGCTGCTCATCTTCCCCGAGGGCACCTGCGTCAACAACGAGTACTGCGTCATGTTCCGCAAGGGCGCCTTRGACCTGGGCGCCACCGTCTGCCCCGTSGCCATCAAGTACAACAAGATCTTCGTCGACGCCTTTTGGAACAGCAAGCGCCAGTCCTTCTCCGCGCACCTCATGAAGATCATGCGCTCGTGGGCCCTCGTCTGCGACGTCTACTTCCTGGAGCCCCAGACGCGGAGGGAGGGCGAGTCGGTCGAGGCGTTTGCGAACCGCGTGCAGGCGCTGATCGCGCAAAAGGCCCGGCTGAAGGTCGCGCCCTGGGACGGCTACCTCAAGTACTACAACCTGGCCGAGAAGCACCCGGACCTGATCGAGAACCAGCGGCGCCAGTACAGCGCCGTCATCAAAAAGTARGCCGAGGASTGAPrototheca moriformis (UTEX 1435) glycerol-3-phosphate acyltransferaseSEQ ID NO: 187MERRTSANPSVGLSSLKHVPLSAVDLPNFESVPKGAASPDVHRQIEELVDKARADRSPASLSLLADVLDISGPLQDAASAMVDDSFLRCFTSTMDEPWNWNFYLFPLWALGVVXRNXILFPLRLLTIVLGTLLFVLAFAVTGFLPKERRLAAEQRCVQFMAQAFVASWTGVIRYHGPRPVAAPNRVWVANHTSMIDYAVLCAYCPFAAIMQLHXGWVGVXQTRYLASLGCLWFNRTQAKDRTLVAKRMREHVASATSTPLLIFPEGTCVNNEYCVMFRKGAXDLGATVCPXAIKYNKIFVDAFWNSKRQSFSAHLMKIMRSWALVCDVYFLEPQTRREGESVEAFANRVQALIAQKARLKVAPWDGYLKYYNLAEKHPDLIENQRRQYSAVIKKXAEXPrototheca moriformis (UTEX 1435) Monoglyceride lipase SEQ ID NO: 188ATGCCCCTCCCGAACGCCATAGAGCTCCCGGGCGCCGATCCCAAATCGAGCTCGGTGTACAGCTTTCAGGGGCACAGCACGAATCTGCAAGTCAACGCCAGGGGCGTCGCKCAGTCCGTGTACGACGARGTCGACCCCGCAACGTACCTGGGGCCCAGAGGGCGGCAGGAGACGGTCGTGAACGCGCAGGGCCTCACGCTGCGGGTTTACTACTGGCCGGCAGAGCAGCCCAAGGCAATCTTGCAGTTTGTGCACGGACATGGCGCGCACGCACTGTTTGAACTCTTGGAMATCGATGAGGCTGGAAAGCCGCCAACGTACGCTGGCTGGGTGGCCAAGCTCAATGCCGCGGGCATCAGCGTGGTCGCCCACGACAATCAGGGCTGCGGGCGCTCAGAGGCGGCGCGGGGCCTGCGCTTCTACATCGAGTCCTTTGACGATTTTGTCAAGGACGTGTTGCTTCTCCGCAGGGAGCTGGTGAAGCAAGAGGGCTTTGACAGCYTGCCGGTYTTCCTGGGGGGCATCTCACTWGGYGGCTGCATAGCGCTCACCTCCCTGCTCGAAGAGCCCGACCARTACCRCGGGCTGTGCCTGCTGGCGCCCATGATCTCGCTSCAAAAGGTGTCCCGCAAGGGCCTYAAYCCCTACCTGCGGCCGCTGGCGGCGCTGCTGAGCCGCGTCGTCCCGTGGGCGCCCATCATCGCGACCGACCGCACCTCCAACTCTGTYCTCCAGCTGCAGTGGGATGCAGACCCACTGACCGCGCACATGAACACGCGCGTCCGCAACGCCAACGAGTACCTCTGCGCGACGGAGCGCGTGACGGCCCGGCTGRGCRCGYTGCAGAAGCCCTTCATCGTCTTCCACTCGGAGAACGACACAATGTGCGACGCGGACGGCTCCAAGCAGCTCTACGCCGAGGCACAGTCCAGCGACAAGACCATCCGCTTTGTCAACAGCATGTGGCACGTGCTGGTCAAGGARCCGGGCAACGAGGAGGTCYTGCAGASTCTGGTGCAGTGGATCCTCGAMCGGGCGTGAPrototheca moriformis (UTEX 1435) Monoglyceride lipase SEQ ID NO: 189MPLPNAIELPGADPKSSSVYSFQGHSTNLQVNARGVXQSVYDXVDPATYLGPRGRQETVVNAQGLTLRVYYWPAEQPKAILQFVHGHGAHALFELLXIDEAGKPPTYAGWVAKLNAAGISVVAHDNQGCGRSEAARGLRFYIESFDDEVKDVLLLRRELVKQEGFDSXPXFLGGISXXGCIALTSLLEEPDXYXGLCLLAPMISXQKVSRKGXXPYLRPLAALLSRVVPWAPIIATDRTSNSXLQLQWDADPLTAHMNTRVRNANEYLCATERVTARLXXXQKPFIVEHSENDTMCDADGSKQLYAEAQSSDKTIRFVNSMWHVLVKXPGNEEVXQXLVQWILXRAPrototheca moriformis (UTEX 1435) acyl-CoA oxidase SEQ ID NO: 190ATGGATGCTCAGAAGCGAGTCCAAGTCTTGTCCGGTCACTTGTCCGAAACTGCGGGCGATGCCGTGGCGGTCTGCCGGGCGGACACCCTGGCCGCGGGGGTGAGCCCCAAGTTCGACCAGGCCCAGGACATGACCGTGTTTCCCCCGGCCAACCACGATGCGCTCTTCCTCAGCGACCTGCTGACTCCCGAGGAAAAGGATGTGCAAATGCGCGTCCGCAAGTTTGCGGAGGAGACCGTTGCGCCCAACGTGATTCCCTACTGGGAAAAGGCCGAGTTCCCGCACGCGCTGCGKCCCGAGTTTGGRAAGCTGGGCATCGGCGGCGGGCACAACAAGGGGCACGGCTGCCCCGGCCTGAGCACCATGGCGGCGGCSTCGGCGGTGGTGGAGCTGGCGCGCGTGGACGGCTCCATGAGCACYTTTTTCCTGGTSCACACCTACCTGGGCGCGATGACGGTGGGCCTGCTGGGCTCSGAGGCGCAGAAGCGGGAGCTGCTGCCCGGCTTCCACRCCTTTGACAAGGTGTGCAGCTGGGCGCTGACGGAGCCGAGCAACGGGTCGGACGCCTCGGCGCTGACCACGACCGCGCGSCGCGTGGAGGGCGGCTGGCTGCTCMGCGGGCGCAAGCGCTGGATCGGCAACGCGACCTTTGCGGACATCATCATCGTCTGGGCGCGCAGCAGCGTGACGGGGCAGGTCAACGCCTTCATCGTGCGCAGGGGCGCGCCGGGGCTGACCACGAGCAAGATCGAGAACAARATCGCGCTGCGCTGCGTGCAGAACGCCGACATCCTGATGGAGGACGTGTTTGTCCCCGACGCGGACCGCCTGCCCGGSGTCAACTCGTTCGCGGACGCGGCGGGCATGCTGGCCATGTCGCGCTGCCTGGTGGCCTGGCAGCCGGTGGGCCTGGTGGTGGGYGTGTACGACATGGTGCTGCGCTACACGCAGCAGCGCCGCCAGTTTGGCGCGCCGCTGGCCGCCTTCCAGCTGGTGCAGGAGCGGCTGGCGCGCATGCTGGGCACCATCCAGGCCATGTACCTCATGTGCGCGCGCCTGAGCAAGCTCTACGACGAGGGCCGCATGACGCACGAGCAGGCCAGCCTGGTCAAGGCCTGGACCACCGCGCGCTCGCGCGAGGTCGTCGCGCTCGGCCGCGAGTGCCTGGGCGGCAACGGCATCGTCGGCGAGTTCCTCGTGGCCAAGGCCTTTGTCGACGCCGAGGCGTATTACACGTATGAAGGGACCTACGACGTCAACGTGCTCGTCGCCGCGCGCGGCCTCACCGGCTACGCCGCCTTCAAGACGCCCGGCCGCGGCGGCAAGAAGGACGMCGCCGCGAAGAAGGAGCACTGA Prototheca moriformis (UTEX 1435) acyl-CoA oxidase SEQ ID NO: 191MDAQKRVQVLSGHLSETAGDAVAVCRADTLAAGVSPKEDQAQDMTVEPPANHDALFLSDLLTPEEKDVQMRVRKFAEETVAPNVIPYWEKAEFPHALXPEFXKLGIGGGHNKGHGCPGLSTMAAXSAVVELARVDGSMSXFFLXHTYLGAMTVGLLGXEAQKRELLPGFHXFDKVCSWALTEPSNGSDASALTTTAXRVEGGWLLXGRKRWIGNATFADIIIVWARSSVTGQVNAFIVRRGAPGLTTSKIENXIALRCVQNADILMEDVFVPDADRLPXVNSFADAAGMLAMSRCLVAWQPVGLVVXVYDMVLRYTQQRRQFGAPLAAFQLVQERLARMLGTIQAMYLMCARLSKLYDEGRMTHEQASLVKAWTTARSREVVALGRECLGGNGIVGEFLVAKAFVDAEAYYTYEGTYDVNVLVAARGLTGYAAFKTPGRGGKKDXAAKKEH Prototheca moriformis (UTEX 1435) enoyl-CoA hydratase SEQ ID NO: 192ATGGCGCCGAGCCCGACCTTCAAGGCACTCAAGGTCTGGATGGACGACGATCTGGTGGGACACATCCAGCTCAATCGTCCTCAAGCCGCCAATTCGATGAATGAGGAGATGTGGACGGAGCTGCCGCAGGCCACAGCGTGGTTGGAGAGCCAGTCTGCGCGCGTCATWCTGGTCACGGGCGCSGGCAAGAACTTTTGCGCGGGCATCGACGTGGCGAGCCTCGGCTCGACCGTGCTCTCCCACGKCGCCGGCTGCGCGGCGCGCTCGGCCTACCGCTTCCGCMCGGGCCTGACCAMSCTGCAGGAGGCCATGACYGCCCTGGAGCGCGTGCGCTGCCCCACCGTGGCGCTGGTGCACGGYCACTGTGTSGGGGCCGGCGTCGACCTCATCACGGCCTGCGACATCCGKTTTGCCACGGCGGACGCCAAGCTSTGCGTCAAGGAGGTCGARCTGGCCGTGATCGCCGACATGGGCACGCTCCAGCGCCTCCCAGGCATCGTGGGSCAKGGCCACGCCCGCGAGCTGTCGCTGACGGCCCGGGTCTTCTCCGGGGAGGAAGCAAAGCAGATGGGTCTCGCCACCGCGGCGCTGCCGAGCCGGGACGAGCTGGAGAGGCACGGCCGCGCGGTCGCGCAGGGCATCGCGGCTAAATCCCCCGTGGCCGTGGCTGGGACCAAGCAGGTGCTGCTCTACCAAAGGGACCACACCGTCGCCGATGGCCTGGACTACGTCAACACCTGGAACGCTGGCATGCTGCACTCGGCTGATTTCGCCGAGGTGTTTGCGGCCATGAAGGAGAGGCGTAAGCCGGTGTTCAGCAAGCTCTGA Prototheca moriformis (UTEX 1435) enoyl-CoA hydratase SEQ ID NO: 193MAPSPTFKALKVWMDDDLVGHIQLNRPQAANSMNEEMWTELPQATAWLESQSARVXLVTGXGKNECAGIDVASLGSTVLSHXAGCAARSAYRFRXGLTXLQEAMXALERVRCPTVALVHXHCXGAGVDLITACDIXFATADAKXCVKEVXLAVIADMGTLQRLPGIVXXGHARELSLTARVFSGEEAKQMGLATAALPSRDELERHGRAVAQGIAAKSPVAVAGTKQVLLYQRDHTVADGLDYVNTWNAGMLHSADFAEVFAAMKERRKPVFSKLPrototheca moriformis (UTEX 1435) mitochondrial acyl carrier protein allele2 SEQ ID NO: 194ATGGCGTTCTTGCAGCGGACGAGCGCGCTGGTGCGCCAGGGTGTCCTGTCTCGCCTCGCCGTGCAGGCCAGCCCCGCCCTGAACAGCGTCCGGGCCTTCGCCTCGGCGTCCTACCTGGACAAGAATGAGGTGACGCACCGCGTGCTGTCGATTGTCAAGAACTTTGACCGCGTCGACGCCGGCAAGGTCACGGACTCTGCCAACTTCCAGTCCGACCTGGGTCTGGACTCACTAGACACCGTGGAGCTGGTCATGGCCCTGGAGGAGGAGTTTGCGATCGAGATCCCGGATGCGGAGGCCGACAAGATCCTCTCCGTCCCGGAGGCGATTTCCTACATTGCCGCGAACCCCATGGCCAAGTAGPrototheca moriformis (UTEX 1435) mitochondrial acyl carrier protein allele2 SEQ ID NO: 195MAFLQRTSALVRQGVLSRLAVQASPALNSVRAFASASYLDKNEVTHRVLSIVKNFDRVDAGKVTDSANFQSDLGLDSLDTVELVMALEEEFAIEIPDAEADKILSVPEAISYIAANPMAKPrototheca moriformis (UTEX 1435) trans-2-enoyl-CoA reductaseSEQ ID NO: 196ATGCCACTGGAAATTCTTGTCAAGACCAGGAATGGTCGACCTGCCTTTTCCAGAGACGGCGGCGCCATTACTGTCGATAGCACGAGTGCGACGGTGCAGGAGGTCAAGGGCCTCATCGCTCGCGCCAAGAAGTTGAGCCCCGCCCGCCTGCGCCTGACGTTGCCCGCGCCGGCCGGCACGCGGCCCACGGTGCTGGAGGACAAGAAGCCCTTGGCCGACTATGGTCTGCACGACGGCGCCAGCCTCGTGCTCAAGGACCTGGGACCTCAGATCGGGTACCAGATGGTCTTTTTCTGGGAGTARTTRGGGCCGCTGGCCATCTACCCCCTCTTCTACTTCCTGCCYTCCTTGATTTACGGAAGGRCGACCGAGCACGTCTTTGCMCAAAAGGCAGCGCTCGCCTTCTGGACCTTRCACTACGSGAAGCGCATCRTGGAGWCCTTTTTCGTGCACAYGTTTGGGCACGCCACGATGCCCGTGCGCAATCTGGTGAAGAACTGCAGCTATTACTGGAGCTTTGGCGCCTTCATCTCCTACTTRGTGAACCACCCGCTCTACMCGGCGCCTCCCGCGGCCCAGACCGCCGTCGCCTTTGTGGCCGCCACCCTCTGCACGCTCTCGAACTTCAAGTGTCATCTGATCCTGAGCAACCTGCGTGCGCCCGGAGGCAGCGGCTATGTCATTCCGCGTGGCTTCCTGTTTGACTACGTCACCTGTGCCAACTACACCGCTGAAATCTGGAGCTGGATTTTCTTCTCCATCGGCACCCAGTGCTTGCCGGCYCTCYTYTTCACCGTRGCTGGYGCCGCGCAAATGGCCATCTGGGCCGGGGGCAAGCATTGCCGTCTGAAAAAGCTCTTCGACGGCAAGGAGGGCCGCGAGCGCTACCCCAAGCGCTACATCATGRTTCCCCCGCTGTACTGAPrototheca moriformis (UTEX 1435) trans-2-enoyl-CoA reductaseSEQ ID NO: 197MPLEILVKTRNGRPAFSRDGGAITVDSTSATVQEVKGLIARAKKLSPARLRLTLPAPAGTRPTVLEDKKPLADYGLHDGASLVLKDLGPQIGYQMVFFWEXXGPLAIYPLEYFLXSLIYGRXTEHVFXQKAALAFWTXHYXKRIXEXFFVHXFGHATMPVRNLVKNCSYYWSFGAFISYXVNHPLYXAPPAAQTAVAFVAATLCTLSNFKCHLILSNLRAPGGSGYVIPRGFLFDYVTCANYTAEIWSWIFFSIGTQCLPXLXFTXAXAAQMAIWAGGKHCRLKKLFDGKEGRERYPKRYIMXPPLYPrototheca moriformis (UTEX 1435) long-chain-fatty-acid-CoA ligaseSEQ ID NO: 198ATGGCTTCCAAGTATCCCCGGCTGACCAAGGTCTCCGATGGCGTGYCTGCCAACGAGGAGGGCGTCGAGTTTGGACCCGAATACAGTTGCACATTCATGAAGGAAAAGACGCTGTCCGTGCCCGGGATCAAGTCTCTGGGCGAGCTCTTTGCGGACTCGGTGTCGCAGCACAAGCGCCGGCCGTGCCTGGGCAAGCGCACCAAGGACGGCTACAGCTGGCGCACGTACGAGCAGACGGGCCGCGAGATCGCGGCCATGGGCGCGGCCTTTGCCGCGCGCGGCCTGGCCCAGCAGTCGCGTGTGGGCGTGTACGGGCCCAACGCGCCCGAGTGGATGATCACGATGCAGGCGTGCAACCGCCAGGGCTACTACTGCGTGCCGCTGTACGACTCKCTGGGCGAGACCGCGGTGGAGTTCATCATCAAGCACGCGGAGGTGAGCGCGGTGGCGGTGGCCGGGCCCAAGCTGGCRGAGCTGGCCAARGCSCTGCCCAACGTGGCGAGCCAGGTCAAGACGGTGGTCTTYTGGGACGASGCCGACCGYGCGGCGGTGGAGGCGGTGCAGGCGCTGGGCGTGGGCGTGTTTGGCTTCGACGAGTTTGTGAAGCAGGGCGAGGCCGCGGAGGCCGTGCCGCCCGCCAGCGTGGAGGGCGAGGACCTGTGCACCATCATGTACACGAGCGGCACGACGGGCGACCCCAAGGGCGTCATGCTCACGCACCGCGCCGTGATCGCGACYGTGCTGAGCCTGSACGCGTTYCTGAAGAGYGTGGACGTGGCGCTGGGCGAGGGCGAYGCCATYYTKTCYTTCCTGCCGCTGGCGCACATCTTYGACCGCGCGGCGGAGGAGCTGATGCTCTACGTGGGCGGCAGCATCGGCTACTGGAGCGGCAACGTCAAGGGCCTGCTGGGCGACATCGCGGCGCTGCGCCCGACGCTCTTCTGCTCCGTGCCGCGCGTGTTTGACCGCATCTACTCCTCGGTCACGGGCAAGGTRGAGAGYGGCGGCTGGCTCAAGCGCACCATGTTCCACCACGCCTTCAAGACCAAGTTYGGGCGGCTGAAGCGCGGCGTGCCGCAGGCCAAGGCCGGCGGCATCTGGGACAAGATCGTGTTCAAGAAGATCAAGGAGGCGCTGGGCGGGCGGTGCAAGATCATCGTCTCGGGCGGCGCGCCGCTGTCGACGCACGTGGAGGAGTTCCTGCGCGTCTGCATGTGCTCGCATGTGGTGCAGGGGTACGGGCTGACCGAGACCTGCGCGGCCAGCTTCATCGCGGAGCCGGCCGACATCCGCCAGATGGGCACGGTCGGCCCGCCGCAGCCGGCGGTCAGCTTCCGCCTGGAGGGCGTGCCGGAGATGAAGTACAGCCCCAGCGCGGACCCCGCGCGTGGCGAGCTGCTCATCCGCGGGCCCGCGCTCTTCTCCGGCTACTACAAGGACCAGGAGAAGACGGACGAGGTGGTCACGCCGGACGGCTGGTTCCACACGGGCGACATCGCGGAGATCACGCCCGCCGGCGCCATCCGCATCATCGACCGCAAGAAGAACATCTTCAAGCTCGCGCAGGGCGAGTACGTGGCCGTSGAGAAGCTGGAGAACACGTACAAGATGTCGCCGGCCGTGGAGCAGATCTGGGTCTACGGCAACAGCTTCGAGAGCGTGCTCGTGGCCGTGGTCGTGCCCAGCGAGGACAAGGTCAAGGCCCACGGCGGCTCCAGCGCCGCGCAGCTCGCCTCGGACGCYGCCTTCAAGAAGGCCGTGCTGGACGACCTCACCGCCGCCGCCAAGGCCGACAAGCTCAAGGGCTTCGAGATGGTCAAGGGCGTCATCATCGAGCCCGAGCCCTTCAGCGTCGAGAACAACCTGCTCACGCCCACCTTCAAGCTCAAGCGGCCGCAGCTGCTCGACCATTACCGCACCGAGATCGACGCGCTCTACGCGAGTCTGAAGAAGTAGPrototheca moriformis (UTEX 1435) long-chain-fatty-acid-CoA ligaseSEQ ID NO: 199MASKYPRLTKVSDGVXANEEGVEFGPEYSCTFMKEKTLSVPGIKSLGELFADSVSQHKRRPCLGKRTKDGYSWRTYEQTGREIAAMGAAFAARGLAQQSRVGVYGPNAPEWMITMQACNRQGYYCVPLYDXLGETAVEFIIKHAEVSAVAVAGPKLXELAXXLPNVASQVKTVVXWDXADXAAVEAVQALGVGVFGFDEFVKQGEAAEAVPPASVEGEDLCTIMYTSGTTGDPKGVMLTHRAVIAXVLSLXAXLKXVDVALGEGXAXXXELPLAHIXDRAAEELMLYVGGSIGYWSGNVKGLLGDIAALRPTLFCSVPRVFDRIYSSVTGKXEXGGWLKRTMEHHAFKTKXGRLKRGVPQAKAGGIWDKIVEKKIKEALGGRCKIIVSGGAPLSTHVEEFLRVCMCSHVVQGYGLTETCAASFIAEPADIRQMGTVGPPQPAVSFRLEGVPEMKYSPSADPARGELLIRGPALFSGYYKDQEKTDEVVTPDGWFHTGDIAEITPAGAIRIIDRKKNIFKLAQGEYVAXEKLENTYKMSPAVEQIWVYGNSFESVLVAVVVPSEDKVKAHGGSSAAQLASDXAFKKAVLDDLTAAAKADKLKGFEMVKGVIIEPEPFSVENNLLTPTFKLKRPQLLDHYRTEIDALYASLKKPrototheca moriformis (UTEX 1435) Enoyl-CoA reductase SEQ ID NO: 200ATGGCGTCCYCYCAGCTCCTGTCCAAGTCCCAGGGTCTGGTCGGTGCCCTGAAGGACGTCAGGGCCGTCCGGGTGYCCCTGCCCCGCGCCGGACCCCYTGCCGCCCCTCGCGCCGATGCCTACTCGAGCTCCAGGGCGCCTGCCTCCATGGTGCTGGCCCCCCAGCGCCGCGGGCTGAGCGTGAAGGCTGCCGCCAGCACCAGCCCTGCCGGCACCGGCTTGCCCATTGATCTGCGGGGCAAGAAGGCCTTTATCGCGGGCGTCGCCGACGACCAGGGTTTTGGCTGGGCCATRGCCAAGCAGCTGGCTGAGGCGGGCGCGGAGATCTCCCTGGGCGTGTGGGTGCCTGCCCTGAACATTTTCGAGAGCAACTACCGCCGCGGCAAGTTTGACGAGAACCGCAAGCTGGCCAACGGCGGCCTGATGGAGTTTGCGCACATCTACCCCATGGATGCGGTCTTCGACTCGCCCGCGGACGTGCCCGAGGACATTGCCAGCAACAAGCGCTACGCCGGGAACAAGGGCTGGACGGTGAGCGAGACGGCCGACAAGGTCGCGGCCGACGTGGGCAAGATCGACATCCTGGTGCACTCGCTGGCCAACGGGCCCGAGGTGCAGAAGCCGCTGCTGGAGACCAGCCGCCGCGGCTACCTCGCCGCSCTGAGCGCCTCCTCCTACTCCCTCATCTCCATGGTCCAGCGCTTTGGCCCGCTCATGAACCCCGGCGGCGCCGTCATCTCGCTGACCTACAACGCCTCCAACCTGGTCATCCCCGGCTACGGCGGCGGCATGAGCACGGCCAAGGCCGCGCTCGAGTCCGACACGCGCGTGCTGGCCTACGAGGCCGGCCGCAAGTACCACGTGCGCGTGAACACCATCAGCGCCGGGCCGCTCGGCTCGCGCGCCGCCAAGGCCATCGGCTTCATCGACGACATGATCCGCTACTCGTACGAGAACGCGCCCATCCAGAAGGAGCTCAGCGCCTACGAGGTCGGCGCSGCCGCCGCCTTCCTCTGCTCCCCGCTCGCCTCGGCCGTCACTGGCCACGTCATGTTTGTCGACAACGGCCTCAACACCATGGGCCTCGCCCTCGACTCCAAGACCCTCGACCYCAGCGAGTGAPrototheca moriformis (UTEX 1435) Enoyl-CoA reductase SEQ ID NO: 201MASXQLLSKSQGLVGALKDVRAVRVXLPRAGPXAAPRADAYSSSRAPASMVLAPQRRGLSVKAAASTSPAGTGLPIDLRGKKAFIAGVADDQGFGWAXAKQLAEAGAEISLGVWVPALNIFESNYRRGKEDENRKLANGGLMEFAHIYPMDAVEDSPADVPEDIASNKRYAGNKGWTVSETADKVAADVGKIDILVHSLANGPEVQKPLLETSRRGYLAXLSASSYSLISMVQRFGPLMNPGGAVISLTYNASNLVIPGYGGGMSTAKAALESDTRVLAYEAGRKYHVRVNTISAGPLGSRAAKAIGFIDDMIRYSYENAPIQKELSAYEVGXAAAFLCSPLASAVTGHVMFVDNGLNTMGLALDSKTLDXSEPrototheca moriformis (UTEX 1435) membrane bound O-acyl transferase domain-containing protein SEQ ID NO: 202ATGGCGCAGAACCTCATCCTCTCCATGCCGTGGGAGGAGTCGGCGTGCTCGTCCCTGGGCCTGTCCCTTTCTGAGCTGCGATTTGTTGTATCATTTTTTGCCTACGTGCTCGTGTCCGCCGTGCTCCGCCACATCCGTGGCACCCGGACCCGGCACTGGTTTGCCCTCGTCACCGGGTTCCTGCTCATCTACTACCCCTTTGGCTCGGGCGTGATGCACGCCTTTGTGTCCTCCACCRTGGTGTACCTGGCKATGGCGGCAGTGCCCTCCCACTGCGGGACSCTGGCCTGGCTCATRGCCTTTCCGTACCTCATCCTCAACCACGTGCTCCAGGCCAGCGGCCTMAGCTGGAAGGAGGGCCAYCTGGATTTCACGGGCGCGCAAATGGTGCTGACGTTGAAGCTGATCGCCGTCGGCGTCTGCTACCAGGACGGCCGGTCCGGCCAGTCGTACGCRAGCCACTACCGCCAGTCCATGCGCCTGCCCYCGCTGCCGGGCCTCCTGGAGTACTACAGCTACTGCTTTGCCTACGGCAACCTCCTGGCCGGCCCCTTTTTCGAGGCGAAAGAGTACTTTGATTTCATGGCSCGGCAGGGRGAGTGGGACGAGGGCGAGCACGGCCGCCTGCCCYGCGGCCTGGGCGCCGGCCTGGTCCGCTTCCTCAAGGGCATCCTGTGCGCCGCGCTGTGGATGCAGCTGGGCAAGCGCTTCAGCGCGAGTCTGCTCGAGTCGGTCTTCTGGACCACGCTGTCCGTCCCGCGACGAATGGCGCTGATCTGGGTCATCGGCTTTGCCGCGCGGCTCAAGTACTACTTTGTCTGGTCGGTGGCCGAGTCTGGGCTCATCCTGTCSGGCCAGTGCTTCGCCGGCAAGACCCAAAAGGGCAAGGCGCAGTGGACGCGGTACATCAACACCAAGATCAGGGAGCTGACGTCGGGCATCTGGCACGGGCTCTTCCCCGGCTACTGGGCCTTTTTCGGGACGAGCGCGCTCATGTTCGAGGCGGCCAAGACCATCTACCGCTACGAGCTGGGCTGGCCGGCCTGGCTGCGCGCGGCGCTGCCCTGGCGCGCCCTCAAGATGGCGCTCACGGCCTTTGTGCTCAACTATGCCGCCATGTCCTTCCTGGTGCTCACCTGGGCCGACACCTGGGCCGTCTGGAAGTCGGTGGGCTTCCTGGGGCACACGCTGCTGCTCCTGATCCTCATAGTCGGCGTCGTGCTGCCGCCCCGCCGCCGACACAAGGGCCAGGCCACCACCGCGCCCGTGGTCACGCCCGCCGCCATCCCGGCCGAGGGAGAGCTGCCMCAYAAGAAGGCAGAGTGAPrototheca moriformis (UTEX 1435) membrane bound O-acyl transferase domain-containing protein SEQ ID NO: 203MAQNLILSMPWEESACSSLGLSLSELRFVVSFFAYVLVSAVLRHIRGTRTRHWFALVTGELLIYYPEGSGVMHAFVSSTXVYLXMAAVPSHCGXLAWLXAFPYLILNHVLQASGXSWKEGXLDFTGAQMVLTLKLIAVGVCYQDGRSGQSYXSHYRQSMRLPXLPGLLEYYSYCFAYGNLLAGPFFEAKEYEDFMXRQXEWDEGEHGRLPXGLGAGLVRELKGILCAALWMQLGKRFSASLLESVFWTTLSVPRRMALIWVIGFAARLKYYFVWSVAESGLILXGQCFAGKTQKGKAQWTRYINTKIRELTSGIWHGLFPGYWAFFGTSALMFEAAKTIYRYELGWPAWLRAALPWRALKMALTAFVLNYAAMSFLVLTWADTWAVWKSVGFLGHTLLLLILIVGVVLPPRRRHKGQATTAPVVTPAAIPAEGELXXKKAEPrototheca moriformis (UTEX 1435) NAD-dependent glycerol-3-phosphatedehydrogenase SEQ ID NO: 204ATGTCCGTCCAACCCATTTCCGAGGAGCTGACCAAGGTGACCGTCATCGGCGCCGGCGCCTGGGGTACCGCCCTCGCCATCCACTGCGCCCGCAAGGGCCATGACACCATGTTGTGGGCCATGGAGCCGCACGCGGTGGAGGAGATCAACACCAAGCACACCAACGAGACTTTCCTGAAGAACGTGCCCCTGCCTGAGTCGCTCAAGGCGACGGGCGACATCAAGGAGGCCGTGGAGCGCGCGGAGATGATCCTGACCGTCATCCCGACTCCCTTCTTGTTCAAGTCGATGAGCGGGATCAAGGAGTACCTGAGGGAGGACCAGTACATCGTGTCCTGCACCAAGGGCATTCTGAACGACACCCTGGAGACCCCCGACGGCATCATCCGCCGCGCCCTGCCCGATAACCTCTGCAAGAGGCTGGCGTTCCTGTCGGGCCCCTCCTTTGCGGCCGAGGTGGGCCGCGACTTCCCCACGGCGGTCACCATCGCGGCCGAGGACCCGGCCGTGGCGCAGCGCGTGCAGCAGCTCATGTCCACCAACCGCTTCCGCTGCTACCGCACCACCGACGTCGTGGGCGTCGAGCTCGCTGGCGCGCTGAAGAACGTGCTGGCCATCGCCTGCGGCATCTCCGACGGCTGCGGCTTTGGCAACAACGGCCGCGCGGCGCTCATCACGCGCGGGCTGGACGAGATCACGCGCATCGCGGTGGCCAGCGGCGCCAACCCGCTGACCCTGGCGGGCCTGGCGGGCGTGGGCGACATCGTGCTCACCTGCTGCGGCGACCTGAGCCGCAACCGCACGGTCGGCCTGCGCCTGGGCAAGGGTGAGAAGCTGGACCACATCGTCAAGACGCTGGGCGCCACGGCCGAGGGCGTGCTCACCTCGCGCGCGGCCTACGACCTCACCAAGAAGCTGGGCATCGACTGCGCCGTCATCCACGGCATCTACCGCGTCGTGAACGAGGAGGCCGAGCCCATGAAGGTCGTGGCCGAGACCATGGCGCGCCCGCTGCGCGCCGAGGTGGCCGAGAGCGTCGCCGAGGCCGCCGTCACCAACGCCCGCAACCTCGCCGAGTAGPrototheca moriformis (UTEX 1435) NAD-dependent glycerol-3-phosphatedehydrogenase SEQ ID NO: 205MSVQPISEELTKVTVIGAGAWGTALAIHCARKGHDTMLWAMEPHAVEEINTKHTNETFLKNVPLPESLKATGDIKEAVERAEMILTVIPTPFLFKSMSGIKEYLREDQYIVSCTKGILNDTLETPDGIIRRALPDNLCKRLAFLSGPSFAAEVGRDEPTAVTIAAEDPAVAQRVQQLMSTNRFRCYRTTDVVGVELAGALKNVLAIACGISDGCGFGNNGRAALITRGLDEITRIAVASGANPLTLAGLAGVGDIVLTCCGDLSRNRTVGLRLGKGEKLDHIVKTLGATAEGVLTSRAAYDLTKKLGIDCAVIHGIYRVVNEEAEPMKVVAETMARPLRAEVAESVAEAAVTNARNLAEPrototheca moriformis Plastidial Acyl-Carrier Protein (ACP) allele 1SEQ ID NO: 206ATGGCGATGTCTATGACCTCCTGCCGCGCCGTCTGCGCCCCTCGCGCCACCCTCCGGGTGCAGGCTCCCCGCGTGGCTGTCCGCCCCTTCCGCGCCCAGCGCATGATCTGCCGCGCTGTGGACAAGGCTAGCGTCCTGAGCGACGTGCGCGTGATCATTGCCGAGCAGCTGGGCACCGATGTCGAGAAGGTCAACGCCGACGCCAAGTTTGCCGACCTGGGTGCCGACTCCCTGGACACCGTTGAGATCATGATGGCTCTGGAGGAGAAGTTTGACCTGCAGCTCGATGAGGAGGGTGCGGAGAAGATCACCACCGTGCAGGAGGCCGCTGACCTCATCTCCGCCCAGATCGGCGCTTGAPrototheca moriformis (UTEX 1435) Plastidial Acyl-Carrier Protein (ACP)allele 1 SEQ ID NO: 207MAMSMTSCRAVCAPRATLRVQAPRVAVRPFRAQRMICRAVDKASVLSDVRVIIAEQLGTDVEKVNADAKFADLGADSLDTVEIMMALEEKFDLQLDEEGAEKITTVQEAADLISAQIGAPrototheca moriformis (UTEX 1435) 3-hydroxyacyl-CoA dehydrataseSEQ ID NO: 208ATGTCGCTGAGGAGCGCCTACCTCACTGTTTATAATGCGTCGCTCGCGCTGGGCTGGGCGTACCTGCTGTGGCTGAGCGTGTCTGTCCTRGCCGCTGGCGGCAGCCTGTGGGACCTGTGGAAGACTGTGGAGGTGCCTCTCAAGGTCGTTCAGACGGCGGCGATCGCGGAAGTTGTGCATGCCAGTGTTGGCATTGTCCGCTCGCCCCCACTCGTCACTGCCCTGCAAGTGGCTTCGCGAGTGTTCCTGGTCTGGGGCGTGGTGAATCTGGCTCCAGAGGTGGCGACCGGCTCGCAGGTGGCCGCCATTCCCATCCCCGGGGTCGGCCGCGTGGGCCTCTCCTTTGCGACCCTGGTCATCGCCTGGGCCCTCAGCGAGATCATCCGCTACGGCCACTTTGCCGCCAAGGAGGCGGGCATTGCCAGCAAGCTGCTGCTCTGGCTTCGCTACACCGGCTTCCTGGTGCTCTACCCGCTGGGCGTCTCCAGCGAGCTGACCATGATCTACCTAGTGGCTCCCTACGTCAAGGAGCGCGGCATCCTGAGCCTGGAGATGCCCAACGCCGCCAACTTTGCCTTTAGCTACTACGCCGCGCTCTGGATCGTCAGCCTCACCTACATTCCCGKMYWWCMKMKGWWRKMCGGGTACATGCTCAAGCAGCGGAAAAAGATGCTTGGTGGTGGCGCCAAGGCCAAGAAACTGGCTTGAPrototheca moriformis (UTEX 1435) 3-hydroxyacyl-CoA dehydrataseSEQ ID NO: 209MSLRSAYLTVYNASLALGWAYLLWLSVSVXAAGGSLWDLWKTVEVPLKVVQTAAIAEVVHASVGIVRSPPLVTALQVASRVELVWGVVNLAPEVATGSQVAAIPIPGVGRVGLSFATLVIAWALSEIIRYGHFAAKEAGIASKLLLWLRYTGFLVLYPLGVSSETMIYLVAPYVKERGILSLEMPNAANFAFSYYAALWIVSLTYIPXXXXXXGYMLKQRKKMLGGGAKAKKLA Prototheca moriformis (UTEX 1435) Ketoacyl-ACP Synthase IISEQ ID NO: 210ATGCAGACCGCGCACCAGCGGCCCCCGACCGAGGGGCACTGCTTCGGTGCGAGGCTGCCCACGGCGTCGAGGCGGGCGGTGCGCCGGGCATGGTCCCGCATCGCGCGCGCGGCGGCCGCGGCCGACGCAAACCCCGCCCGCCCTGAGCGCCGCGTGGTCATCACGGGCCAGGGCGTGGTGACCAGCCTGGGCCAGACGATCGAGCAGTTTTACAGCAGCCTGCTGGAGGGCGTGAGCGGCATCTCGCAGATCCAAAAGTTTGACACCACGGGCTACACGACGACGATCGCGGGCGAGATCAAGTCGCTGCAGCTGGACCCGTACGTGCCCAAGCGCTGGGCCAAGCGCGTGGACGACGTCATCAAGTACGTCTACATCGCGGGCAAGCAGGCGCTGGAGAACGCGGGGCTGCCGATCGAGGCGGCGGGGCTGGCGGGCGCGGGGCTGGACCCGGCGCTGTGCGGCGTGCTCATCGGCACCGCCATGGCGGGCATGACGTCCTTCGCGGCGGGCGTGGAGGCGCTGACGCGCGGCGGCGTGCGCAAGATGAACCCCTTTTGCATCCCCTTCTCCATCTCCAACATGGGCGGCGCGATGCTGGCGATGGACATCGGCTTCATGGGCCCCAACTACTCCGTCTCCACAGCCTGCGCGACGTCCAACTACGCATTTGTGAACGCGGCCAACCACATCCGCAAGGGCGACGCGGACGTCATGGTCGTGGGCGGCACCGAGGCCTCCATCGTGCCCGTGGGCCTGGGCGGCTTTGTGGCCTGCCGCGCGCTGTCCACGCGCAACGACGAGCCCAAGCGCGCGAGCCGGCCGTGGGACGAGGGCCGCGACGGCTTTGTGATGGGCGAGGGCGCGGCCGTGCTGGTCATGGAGTCGCTGGAGCACGCGCAGAAGCGTGGCGCGACCATCCTGGGCGAGTACCTGGGGGGCGCCATGACCTGCGACGCGCACCACATGACGGACCCGCACCCCGAGGGCCTGGGCGTGAGCACCTGCATCCGCCTGGCGCTCGAGGACGCCGGCGTCTCGCCCGACGAGGTCAACTACGTCAACGCGCACGCCACCTCCACCCTGGTGGGCGACAAGGCCGAGGTGCGCGCGGTCAAGTCGGTCTTTGGCGACATGAAGGGTATCAAGATGAACGCCACCAAGAGTATGATCGGGCACTGCCTGGGCGCCGCCGGCGGCATGGAGGCCGTCGCCACGCTCATGGCCATCCGCACCGGCTGGGTGCACCCCACCATCAACCACGACAACCCCATCGCCGAGGTCGATGGCCTGGACGTCGTCGCCAACGCCAAGGCCCAGCACGACATCAACGTCGCCATCTCCAACTCCTTCGGCTTTGGCGGGCACAACTCCGTCGTCGCCTTTGCGCCCTTCCGCGAGTAGPrototheca moriformis (UTEX 1435) Ketoacyl-ACP Synthase IISEQ ID NO: 211MQTAHQRPPTEGHCFGARLPTASRRAVRRAWSRIARAAAAADANPARPERRVVITGQGVVTSLGQTIEQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALENAGLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMDIGFMGPNYSVSTACATSNYAFVNAANHIRKGDADVMVVGGTEASIVPVGLGGEVACRALSTRNDEPKRASRPWDEGRDGFVMGEGAAVLVMESLEHAQKRGATILGEYLGGAMTCDAHHMTDPHPEGLGVSTCIRLALEDAGVSPDEVNYVNAHATSTLVGDKAEVRAVKSVFGDMKGIKMNATKSMIGHCLGAAGGMEAVATLMAIRTGWVHPTINHDNPIAEVDGLDVVANAKAQHDINVAISNSFGFGGHNSVVAFAPFRE Prototheca moriformis (UTEX 1435) sterol 14 desaturaseSEQ ID NO: 212ATGGACGCCTTTGGCGAGCTCATCATCCTGACCGCCTCGCGCACGCTGCTGGGCCGCGAGGTGCGCGAGTCCATGTTCCGCGAGGTGGCCGACCTCTTCCACGACCTGGACGACGGCATGCGCCCGCTGAGCGTGCTCTTCCCCTACCTGCCGACCGCCTACCACAGGCGCCGCGACGTCGCGCGCCGGCGCCTGCAGGACATCTTCAAGCGCGTCATCGCCGCGCGCCGCGCCTCGGGCGTCAAGGAGGACGACGTGCTGCAGGCGCTGATCGACGCGCGCTACCGCAAGGTCTACGACGGCCGCGCCACGACGGACGAGGAGATCACGGGCCTCCTCATCGCGCTGCTCTTCGCCGGCCAGCACACCAGCTCCGTGACCTCCACCTGGACGGGYCTCTACATGATGMCSGGCGACCAGCGCTACTTCAYGGTGGTCGAGGAGGAGCAGCGCCGCGTCGTCGCCGAGCACGGCGACGCGCTCGACTTTGACGTGCTCAACGGCATGGACCGCCTGCACCTGGCCATCATGGAGGCCCTGCGCCTGCAGCCGCCCCTCGTCCTCCTCATGCGCTACGCCAAGGAGCCCTTCGAGGTCGTCACCTCCGACGGCAAGGCCTTCACCGTGCCCAAGGGCGACATTGTGGCGACCTCGCCCTCCTTCTCCCACCGCCTCAAAAACGTGTTCCGGGATCCCAACGACTTCCAYCCCGAYCGCTTTGAAGCGCCCCGCGACGAGGACAAGGCCGTGCCCTTCTCCTACATCGGRTTTGGCGGCGGGCGGCACGGGTGCATGGGCTCGCAGTTCGCCTACCTCCAGATCAAGACCATCTGGAGCGTGCTCCTCCGCAACTTCACCTTTGAGCTGGTGGACCCCTTCCCGGAGCCGGACTGGGCCTCCATGGTCATCGGGCCCAAGCCCTGCCGCGTGCGCTACACGCGCCGAAAGACGCCCCTGGCCTGAPrototheca moriformis (UTEX 1435) sterol 14 desaturase SEQ ID NO: 213MDAFGELIILTASRTLLGREVRESMFREVADLEHDLDDGMRPLSVLEPYLPTAYHRRRDVARRRLQDIFKRVIAARRASGVKEDDVLQALIDARYRKVYDGRATTDEEITGLLIALLFAGQHTSSVTSTWTXLYMMXGDQRYFXVVEEEQRRVVAEHGDALDFDVLNGMDRLHLAIMEALRLQPPLVLLMRYAKEPFEVVTSDGKAFTVPKGDIVATSPSFSHRLKNVERDPNDEXPXRFEAPRDEDKAVPFSYIXFGGGRHGCMGSQFAYLQIKTIWSVLLRNFTFELVDPFPEPDWASMVIGPKPCRVRYTRRKTPLAPrototheca moriformis (UTEX 1435) Ketoacyl-ACP Synthase IIISEQ ID NO: 214ATGCGCATACTGGGCAGCGGTTCCTCCACCCCTTCCACGGTGCTGAGCAACGCCGATCTGGAGCAGCTGGTGGAGACGAACGACGAGTGGATCGTTGCCCGGACTGGCATCCGGCGCCGGCACATCCTGGGCCCGGGGGAGACGCTCACCCACCACTGCACCCTGGCCTGCCAGCGCGCGCTAGAGATGGCGGGCGTGGACGCCAAGGACGTGGACCTCATCCTGCTGGCCACGTCCAGCCCGGACGACTCCTTTGGCAGCGCCTGTGCTGTGCAGGCCGAGCTCGGGGCCAAGAGCGCGGCCGCCTACGACCTCACCGCCGCCTGCTCCGGCTTTGTCATGAGCACGGTCACCGCCACCCAATTCTTGAGAACGGGGGTGTACAAGAACATCCTGGTCATCGGCGCGGACGCCCTGTCCCGCTACATCGACTGGCGGGACCGGAGCACGTGCATCCTGTTTGGCGACGGCGCCGGCGCTGTCCTCCTCCAGCGCGACGATCGCGAGGGTGAGGACTCCCTTCTCGGGTTCGACATGCACTCTGATGGGCACGGCCAGAAGCACCTCCACGCCGGCTTCATGGGCTGCGGCAACAAGGCCTTCTCGGAGCAGCCCTCCAGCGCCGCCGCTTTCGGCAACATGTACATGAACGGCAGCGAGGTCTTCAAGTTTGCCGTGCGAGCCGTGCCGCGGGTGGTGGAGGCCTCGCTGAAAATGGCGGGGCTGAGCGTGTCCGACATCGACTGGCTGGTCCTGCACCAGGCCAACCAACGAATTTTGACGGCTGCCGCCGATCGGCTGGGTGTCCCCGCTGAGAAAGTGGTGTCCAATGTGGCCGAGTACGGCAACACGTCGGCCGCCTCCATCCCCCTCGTGCTGGACGAATCGGTCCGGCAGGGCCTGATCAAGCCGGGCGACATCCTGGGCATGGCAGGGTTTGGGGCTGGCCTGTCCTGGGCGGGAGCCATCCTCCGATGGGGCTAG Prototheca moriformis (UTEX 1435) Ketoacyl-ACP Synthase IIISEQ ID NO: 215MRILGSGSSTPSTVLSNADLEQLVETNDEWIVARTGIRRRHILGPGETLTHHCTLACQRALEMAGVDAKDVDLILLATSSPDDSFGSACAVQAELGAKSAAAYDLTAACSGFVMSTVTATQFLRTGVYKNILVIGADALSRYIDWRDRSTCILFGDGAGAVLLQRDDREGEDSLLGEDMHSDGHGQKHLHAGFMGCGNKAFSEQPSSAAAFGNMYMNGSEVEKFAVRAVPRVVEASLKMAGLSVSDIDWLVLHQANQRILTAAADRLGVPAEKVVSNVAEYGNTSAASIPLVLDESVRQGLIKPGDILGMAGFGAGLSWAGAILRWGPrototheca moriformis (UTEX 1435) acyl transferase SEQ ID NO: 216ATGAAGGAGCAAGTGGTTATGCCCCCCCCTGAGGCCCCCACGAATGGGGACCTTGCGGCCCCCTTTGTGCGGAAGGACCGGTTCGGGGAGTATGGCATGGCCCCGCAGCCCCCGATGGTGAAGGTGGTGCTGGCACTCGAGGCCTTGGTGCTCTTGCCGCTGCGCGCGGTGAGCGTGCTCATCCTCGTCGTGCTGTACTGGCTCATTTGCAATGCTTCGGTCGCACTCCCGCCCAAGTACTGCGCGGCCGTCACGGTCACCGCTGGGCGCTTGGTCTGCCGGTGGGCGCTCTTCTGCTTCGGATTCCACTACATCAAGTGGGTGAACCTGGCGGGCGCGGAGGAGGGCCCCCGCCCGGGCGGCATTGTTAGCAACCACTGCAGCTACCTGGACATCCTGCTGCACATGTCCGATTCCTTCCCCGCCTTTGTGGCGCGCCAGTCGACGGCCAAGCTGCCCTTTATCGGCATCATCAGCCAAATTATGAGCTGCCTCTACGTGAACCGCGACCGCTCGGGGCCCAACCACGTGGGTGTGGCCGACCTGGTGAAGCAGCGCATGCAGGACGAGGCCGAGGGGAAGACCCCGCCCGAGTACCGGCCGCTGCTCCTCTTCCCCGAGGGCACCACCTCCAACGGCGACTACCTGCTTCCCTTCAAGACCGGCGCCTTCCTGGCCGGGGTGCCCGTCCAGCCCGTCGTCCTCCACTACCACAGGGGAACCCTGTGGCCCACGTGGGAGACGATTCCGGCCAAGTGGCACATCTTCCTGATGCTCTGCCACCCCCGCCACAAAGTGACCGTGATGAAGTTGCCCGTGTACGTCCCCAATGAGGAGGAAAAGGCCGACCCCAAGCTGTACGCCCAAAACGTCCGCAAAGCCATGATGGAGGTCGCCGGGACCAAGGACACGACGGCGGTGTTTGAGGACAAGATGCGCTACCTGAACTCCCTGAAGAGAAAGTACGGCAAGCCTGTGCCTAAGAAAATTGAGTGA Prototheca moriformis (UTEX 1435) acyl transferaseSEQ ID NO: 217MKEQVVMPPPEAPTNGDLAAPFVRKDRFGEYGMAPQPPMVKVVLALEALVLLPLRAVSVLILVVLYWLICNASVALPPKYCAAVTVTAGRLVCRWALFCFGEHYIKWVNLAGAEEGPRPGGIVSNHCSYLDILLHMSDSFPAFVARQSTAKLPFIGIISQIMSCLYVNRDRSGPNHVGVADLVKQRMQDEAEGKTPPEYRPLLLFPEGTTSNGDYLLPFKTGAFLAGVPVQPVVLHYHRGTLWPTWETIPAKWHIFLMLCHPRHKVTVMKLPVYVPNEEEKADPKLYAQNVRKAMMEVAGTKDTTAVFEDKMRYLNSLKRKYGKPVPKKIEPrototheca moriformis ENR1-1(Enoyl-ACP Reductase) 1 cDNA sequence; version1 SEQ ID NO: 218ATGGCGTCCGCACAGCTCCTGTCCAAGTCCCAGGGTCTGGTCGGTGCCCTGAAGGACGTCAGGGCCGTCCGGGTGACCCTGCCCCGCGCCGGACCCCGTGCCGCCCCTCGCGCCGATGCCTACTCGAGCTCCAGGGCGCCTGCCTCCATGGTGCTGGCCCCCCAGCGCCGCGGGCTGAGCGTGAAGGCTGCCGCCAGCACCAGCCCTGCCGGCACCGGCTTGCCCATTGATCTGCGGGGCAAGAAGGCCTTTATCGCGGGCGTCGCCGACGACCAGGGTTTTGGCTGGGCCATCGCCAAGCAGCTGGCTGAGGCGGGCGCGGAGATCTCCCTGGGCGTGTGGGTGCCTGCCCTGAACATTTTCGAGAGCAACTACCGCCGCGGCAAGTTTGACGAGAACCGCAAGCTGGCCAACGGCGGCCTGATGGAGTTTGCGCACATCTACCCCATGGATGCGGTCTTCGACTCGCCCGCGGACGTGCCCGAGGACATTGCCAGCAACAAGCGCTACGCCGGGAACAAGGGCTGGACGGTGAGCGAGACGGCCGACAAGGTCGCGGCCGACGTGGGCAAGATCGACATCCTGGTGCACTCGCTGGCCAACGGGCCCGAGGTGCAGAAGCCGCTGCTGGAGACCAGCCGCCGCGGCTACCTCGCCGCGCTGAGCGCCTCCTCCTACTCCCTCATCTCCATGGTCCAGCGCTTTGGCCCGCTCATGAACCCCGGCGGCGCCGTCATCTCGCTGACCTACAACGCCTCCAACCTGGTCATCCCCGGCTACGGCGGCGGCATGAGCACGGCCAAGGCCGCGCTCGAGTCCGACACGCGCGTGCTGGCCTACGAGGCCGGCCGCAAGTACCACGTGCGCGTGAACACCATCAGCGCCGGGCCGCTCGGCTCGCGCGCCGCCAAGGCCATCGGCTTCATCGACGACATGATCCGCTACTCGTACGAGAACGCGCCCATCCAGAAGGAGCTCAGCGCCTACGAGGTCGGCGCGGCCGCCGCCTTCCTCTGCTCCCCGCTCGCCTCGGCCGTCACTGGCCACGTCATGTTTGTCGACAACGGCCTCAACACCATGGGCCTCGCCCTCGACTCCAAGACCCTCGACCACAGCGAGTGAPrototheca moriformis ENR1-1(Enoyl-ACP Reductase) 1 cDNA sequence; version2 SEQ ID NO: 219ATGGCGTCCGCACAGCTCCTGTCCAAGTCCCAGGGTCTGGTCGGTGCCCTGAAGGACGTCAGGGCCGTCCGGGTGACCCTGCCCCGCGCCGGACCCCGTGCCGCCCCTCGCGCCGATGCCTACTCGAGCTCCAGGGCGCCTGCCTCCATGGTGCTGGCCCCCCAGCGCCGCGGGCTGAGCGTGAAGGCTGCCGCCAGCACCAGCCCTGCCGGCACCGGCTTGCCCATTGATCTGCGGGGCAAGAAGGCCTTTATCGCGGGCGTCGCCGACGACCAGGGTTTTGGCTGGGCCATCGCCAAGCAGCTGGCTGAGGCGGGCGCGGAGATCTCCCTGGGCGTGTGGGTGCCTGCCCTGAACATTTTCGAGAGCAACTACCGCCGCGGCAAGTTTGACGAGAACCGCAAGCTGGCCAACGGCGGCCTGATGGAGTTTGCGCACATCTACCCCATGGATGCGGTCTTCGACTCGCCCGCGGACGTGCCCGAGGACATTGCCAGCAACAAGCGCTACGCCGGGAACAAGGGCTGGACGGTGAGCGAGACGGCCGACAAGGTCGCGGCCGACGTGGGCAAGATCGACATCCTGGTGCACTCGCTGGCCAACGGGCCCGAGGTGCAGAAGCCGCTGCTGGAGACCAGCCGCCGCGGCTACCTCGCCGCGCTGAGCGCCTCCTCCTACTCCCTCATCTCCATGGTCCAGCGCTTTGGCCCGCTCATGAACCCCGGCGGCGCCGTCATCTCGCTGACCTACAACGCCTCCAACCTGGTCATCCCCGGCTACGGCGGCGGCATGAGCACGGCCAAGGCCGCGCTCGAGTCCGACACGCGCGTGCTGGCCTACGAGGCCGGCCGCAAGTACCACGTGCGCGTGAACACCATCAGCGCCGGGCCGCTCGGCTCGCGCGCCGCCAAGGCCATCGGCTTCATCGACGACATGATCCGCTACTCGTACGAGAACGCGCCCATCCAGAAGGAGCTCAGCGCCTACGAGGTCGGCGCGGCCGCCGCCTTCCTCTGCTCCCCGCTCGCCTCGGCCGTCACTGGCCACGTCATGTTTGTCGACAACGGCCTCAACACCATGGGCCTCGCCCTCGACTCCAAGACCCTCGACCGCAGCGAGTGAPrototheca moriformis ENR1-1(Enoyl-ACP Reductase) 1 Protein sequence;version 1 SEQ ID NO: 220MASAQLLSKSQGLVGALKDVRAVRVTLPRAGPRAAPRADAYSSSRAPASMVLAPQRRGLSVKAAASTSPAGTGLPIDLRGKKAFIAGVADDQGFGWAIAKQLAEAGAEISLGVWVPALNIFESNYRRGKEDENRKLANGGLMEFAHIYPMDAVEDSPADVPEDIASNKRYAGNKGWTVSETADKVAADVGKIDILVHSLANGPEVQKPLLETSRRGYLAALSASSYSLISMVQRFGPLMNPGGAVISLTYNASNLVIPGYGGGMSTAKAALESDTRVLAYEAGRKYHVRVNTISAGPLGSRAAKAIGFIDDMIRYSYENAPIQKELSAYEVGAAAAFLCSPLASAVTGHVMFVDNGLNTMGLALDSKTLDHSE*Prototheca moriformis ENR1-1(Enoyl-ACP Reductase) 1 Protein sequence;version 2 SEQ ID NO: 221MASAQLLSKSQGLVGALKDVRAVRVTLPRAGPRAAPRADAYSSSRAPASMVLAPQRRGLSVKAAASTSPAGTGLPIDLRGKKAFIAGVADDQGFGWAIAKQLAEAGAEISLGVWVPALNIFESNYRRGKEDENRKLANGGLMEFAHIYPMDAVEDSPADVPEDIASNKRYAGNKGWTVSETADKVAADVGKIDILVHSLANGPEVQKPLLETSRRGYLAALSASSYSLISMVQRFGPLMNPGGAVISLTYNASNLVIPGYGGGMSTAKAALESDTRVLAYEAGRKYHVRVNTISAGPLGSRAAKAIGFIDDMIRYSYENAPIQKELSAYEVGAAAAFLCSPLASAVTGHVMFVDNGLNTMGLALDSKTLDRSE*Prototheca moriformis Heteromeric acetyl-CoA carboxylase b-CT subunitSEQ ID NO: 222ATGGCACTTCCACTTCCGCCACTCCCATCACTTCCAAAACTTCCAAAAATTCAATTTCCTTTTTTTTCTTGGTTAGATTGGGGAAAAGATAAACGACTTGAGAAAAAAAAAGAAATTGAAAATAAAATAGAGCAACAAATACCGAATCTAATAAAGTTAAGTGCACTTAAATATAAGAGAGATGTTAAAGCTGAAAAAGATTTAGGTTTATGGACTCGTTGTGAAAATTGTGGTGTTATTATATACATAAAACATTTACAAGTAAACAAAAAAGTTTGTATAGGATGTAATTATCATATATTAATGAGCAGTTTTGAACGAATTGCTCGCTTTCTTGACATAGAAAGTTGGACTCCATTAAATGAAACAATGTCTTCAGGTAATCCTTTAGGATTTAGTGATCAAAAATCATATACAGAAAGATTAATAGAGTCTCAAGAATTAACAGGATTACAGGATGCAGTTCAAACAGGAACTGGCGTTATAGAAGGTATTCCTTTAGCACTCGGAATGATGGATTTCCATTTTATGGGTGGTAGTATGGGTTCTGTTGTTGGAGAAAAATTAACTCGTTTAATTGAATATGCAACAATTAAAGGCCTTTCTTTAGTAATTTTTTGTGCTTCTGGTGGTGCTCGTATGCAAGAAGGTATTTTAAGTTTAATGCAAATGGCTAAAATTTCTGCTGCATTAAATCGTCATCAAAATATAGCAAAACTTTTATATATCCCAGTTTTAACATCACCTACTACAGGAGGAGTTACTGCAAGTTTTGCGATGCTAGGAGATTTAATTTATGCAGAACCTCGTGCACTTATTGGTTTTGCTGGACGACGAGTTATTTCTCAAACACTTCAAGAACAATTACCAAAAGATTTTCAAACAGCTGAATATTTATTACATCATGGATTAATTGATTTAATTATTTCAAGATTTTTCTTAAAACAAGCATTAGCTGAAACAGTTATTTTATTTCAACAAGCTCCTTCAAAAGAAATAGGATCTATTGAATTCCGTGAACGTGTTACTTTAACAAAAGTTGAAGAAGAATTATTAAGACGTTCAATAATCGAGAATGAAAACTCTTTTAAAAAAATGTTAGAATCGTTTTTATCTTTTATTCCAGGAAGAAAAAAAGCAATTTCAACAGATTTAGCATTTAATGAAGTTCTTGATGAAGCTTTTGATAATTCATTAAATGCTTCTATAACAAAAGACTTCAGTTATATGCTTTCTTCAACTAAATTAATTGAAGCTTAAPrototheca moriformis Heteromeric acetyl-CoA carboxylase b-CT subunitSEQ ID NO: 223MALPLPPLPSLPKLPKIQFPFFSWLDWGKDKRLEKKKEIENKIEQQIPNLIKLSALKYKRDVKAEKDLGLWTRCENCGVIIYIKHLQVNKKVCIGCNYHILMSSFERIARFLDIESWTPLNETMSSGNPLGESDQKSYTERLIESQELTGLQDAVQTGTGVIEGIPLALGMMDFHFMGGSMGSVVGEKLTRLIEYATIKGLSLVIFCASGGARMQEGILSLMQMAKISAALNRHQNIAKLLYIPVLTSPTTGGVTASFAMLGDLIYAEPRALIGFAGRRVISQTLQEQLPKDFQTAEYLLHHGLIDLIISRFFLKQALAETVILFQQAPSKEIGSIEFRERVTLTKVEEELLRRSIIENENSFKKMLESELSFIPGRKKAISTDLAFNEVLDEAFDNSLNASITKDFSYMLSSTKLIEA*Prototheca moriformis Glycerol-3-phosphate acyltransferase (GPAT)nucleotideSEQ ID NO: 224TCGGCACCTGGATGAGGGCCTGGACGACATGCCAGCCCATGTCAAGGTTGCTGCCCGTGGGCATGACAGTGCAAGGGAAATAGGTCATCTTTCACACGCTCACTATAATCAATGAAACATCTACTTTCATCATATTGCAGTCTTCTTTGATGGGAGTCGGTCTAACAATCCCCATCAGTCACGGCCGGCTGGCACTAGGTACCTGGCAAGGAGTCTACTTGAATGAGCACAGGGATTGCGGGGGCGCACGCCATCTGATTGTGACGCTGCAGGGGTGCTCTTCCTGATGAGGGTTGGGCCCCATGGCAGCTCGGCGCAATCTGGATCTGATCCCATGCTGGGGCTTTAAAACTGCGTTCCGACATGCGACAGGGACAATGCGCGCCCCCACTGACGGCCTTTCATAGCCGTCAGCATAAACAAGATACCACTTGACAGAGTTGCGGGTTGCAAGGGGCTCAAGGCCTCTGGGGGCGCGCCGCTCTTCGCCATGCAAACCTTGGCTAGCACCCGCACCGTGCCCGCGGCGTCGGGCACCGATCGGGCCGCTCATGGGGCGATCAGATATCATGCCCAGCGCATTCAATCAAGGTTTGCGGCTTCAAGGAGGGGCGCCCGGGCTCTGGGCCCCCCCCGGGCTGTGGCGTCTGCCAAGACGTCCCCTGGGGAGCTGCGCCTGCCCTTTAGCCACGTCCGCTCGGAGGAAGAGCTGTTCGCCATTTTGCGGTCCGGCGTGGCGCATGGCCAGCTGCCCGCGTCGATTCTGAACCGCGCCTCGGAGCTCATGGAGTCGTACACGGCATCCATGAAGGCCGGCGGCGTTAGCGACCCGAGCGCTTACACGGTCAAGATAATGGCGGAGCACTTTGGTCTGTCCATGCTGCAAATGGCCCATCGGCACAAGTTTGATTCGGCGCACAAGCGGGTGCTGGAGCCGTACGACTACTACTCCTTTGGCCAGCGCTACATCCAGGGCCTCATCGACATGCAGCGCTCCTGCCTGGGCCACGTGGACCGCTTCGCCGAGATCCAAGCGCAGCTGGACAGAGGCGAGAACGTGGTGCTGCTCGCAAACCACCAGACGGAGGCCGACCCCGCGGTGTGGGCGCTGCTGCTGGAGTCGCGCTTCCCGAAGCTGGCGACGGAGGTGGCGTACGTGGCGGGCGACCGCGTGATCACCGACCCGCTCTGCATCCCCTTTTCGCTGGGCCGCAACCTCTACTGCGTGCACTCCAAGAAGCACCTGGACGACGTCCCGGAGCTCAAGGCGGAGAAGGCCGCGACAAACCGCCAGACCCTGCGCGTCATGGCCCGCGACCTGGCCGCGGGCGGGGTGCTCCTCTGGGTTGCGCCCTCGGGCGGCCGGGACCGCGCCGTCTGCCCGAGCACGGGCGAGACCCTGCCCGACCCCTTTGACCCCGCGGTCGTGGAGCTCATGCGGGCCCTGCTCGCCAAGGCCGGCCGGCCGGGGCACCTCTGGCCCCTGGCCATGTACTCTGCCAGGGTGATGCCCCCGCCGACGACCATTGAGAAGGACATTGGCGAACGGCGGGTCGTCGGCTTTGCCGGCTGCGGTCTCTCCCCCGGACCCGAACTCGATGTGGCAGCCCTGGTGCCTCCCAACGTGACGGACCGTGCGCAGCGGCAGCAGCTGCTCTCGGATGCGGCGTTTGAGTCCATCTCTCACGAGTACGACCGGCTGTTGAAAGCGGTGCATGGGCAGCTGAGTGGTGCGGAAGCCGAGGCGTACACCCAGCCCAAGCTCGAGTCCTAGTTGCTGCAAGTTTCCCTCGCGCTGGGCGGCGGTGTGCCACCCAAGCGTTGGGGACCTGGGTTTTCCAGGCTTGGCCTCCCACCGCCGGTCAAGACTGGCTCTCGACCTCGGCGACCCCGGCTTTCCCTGTCGTCCAATGTGTGCGGCACTCTGAGCGACGACCACCACCCAGTGATGCCTCAGTTTTTCACTAGCTCTTGCGCATAGATAGCCCCCTCTGAAACCTGTTCATGCCTTCACCCTCTTCCAAGAGTCACCTGCAACCTTTTTGGCACCAAAAAAAAPrototheca moriformis Glycerol-3-phosphate acyltransferase (GPAT), proteinSEQ ID NO: 225MQTLASTRTVPAASGTDRAAHGAIRYHAQRIQSRFAASRRGARALGPPRAVASAKTSPGELRLPFSHVRSEEELFAILRSGVAHGQLPASILNRASELMESYTASMKAGGVSDPSAYTVKIMAEHFGLSMLQMAHRHKEDSAHKRVLEPYDYYSFGQRYIQGLIDMQRSCLGHVDRFAEIQAQLDRGENVVLLANHQTEADPAVWALLLESRFPKLATEVAYVAGDRVITDPLCIPFSLGRNLYCVHSKKHLDDVPELKAEKAATNRQTLRVMARDLAAGGVLLWVAPSGGRDRAVCPSTGETLPDPFDPAVVELMRALLAKAGRPGHLWPLAMYSARVMPPPTTIEKDIGERRVVGFAGCGLSPGPELDVAALVPPNVTDRAQRQQLLSDAAFESISHEYDRLLKAVHGQLSGAEAEAYTQPKLES*Prototheca moriformis PAP, nucleotide SEQ ID NO: 226GGCCGGCGTGAAGCCCTCCAAGGGCAGGCTGGTCCCCGTGGCCAACCCGGCCTGGAGCGCGCTCATCAACTCCCACTGGCACCAGTACCTTGCCATGGTTCTGGGCTACCTCGTGTGCCTCTACAGCGACCGGGCCACGCCCTTTCGCCGCGCCATCTACCACGAGACCGACGCCGAGCTCTGGAGGTATAGCTACCCGCTGCTCCCGAACCAGGTCCCCGCCCAGGCGGTGGTGCTCCTCTGCTTCACCGCCCCCTTTGCCGTCATCCTGCCCTTTTACTTTGCGGGCCGCATCTCGCGCATCGTGGCGCACCACGGCCTGCTCCAGGCCTTTGCGGCCGTCGTGATCACTGGCCTTATCACCAACCTCATCAAGCTGAATGTGGCGCGTCCCCGGCCCGACTTTGTGGCGCGGTGCTGGCCCCACGGCGCCGCGCGCGCCTTCACCGACGTGGGCGTGCCCATCTGCGCGCCCGACGCGATCGACTGGGTGGAGGGCCTCAAGTCCTTCCCCTCCGGGCACACCTCCTGGGCCGCCTCGGGCATGGCCTACCTCACGCTCTGGCTGGTGGGCGCGCTGCGCGTTTTCTCCGGCGCGGCGCGGCCGACCAGCATCGCGGCCGCGCTGTGCCCGCTGTGCCTGGCCGCCTGGATCGGCATGACGCGCATCCAGGACCGGTGGCACCACACGGAGGACGTCATGGTCGGCTTCAGCCTGGGCAGCACGATTGCCTACCTCTGCTACCGCGCGGTGCACTGCCCCGTCAACGGACCGCACGCGGGCGCGCTCACCGCGCTGGCCCTGCCGGACGAGGGGCCCGGCGCCGGGCCCAGCATCAGCCGCGAGCCGAGCTTTGCGCAGATGGCGGCCGCGGCCGGCGTGGGCGTGCCCCTGCAGCCCGTGCGCTACAACCCCCTCGTGGAAACCACGCCCGAAGAGACCGTCTAGGACGGGGGGTGGGGCGAGAGGATGTCAAGGCTGCTGTCGGTGTGCTGTTTCTTTATGCCCTGGCCAGTCTGCCAGTGCATGCACCACAATTTTGACGACTGTACCGCTAAACACGAGCTCCCCCTGCCAGGTGACCTCGATGCGTGTGGCAAAAGAAACGTGTATTTTGTGCTTCCCGCCCGTGCCGTTGCGCGGGCAGGTTTTTCTACCGCCTCCTTCTGGAATGTGTCGGGCCGCTCGATCGAGGGCAGTGGCCCCTGCCATTCTTCTTATTTTTAGAATTTGATCCTCAGPrototheca moriformis PAP, protein SEQ ID NO: 227MVLGYLVCLYSDRATPERRAIYHETDAELWRYSYPLLPNQVPAQAVVLLCETAPFAVILPFYFAGRISRIVAHHGLLQAFAAVVITGLITNLIKLNVARPRPDEVARCWPHGAARAFTDVGVPICAPDAIDWVEGLKSEPSGHTSWAASGMAYLTLWLVGALRVFSGAARPTSIAAALCPLCLAAWIGMTRIQDRWHHTEDVMVGFSLGSTIAYLCYRAVHCPVNGPHAGALTALALPDEGPGAGPSISREPSFAQMAAAAGVGVPLQPVRYNPLVETTPEETV*Prototheca moriformis DGAT1-1, nucleotide SEQ ID NO: 228CTGGAGCTGTGCTGGCCGCTGCTGGCGCTGCTGGCGCTGGCCGACGCCGCGGCCAGCGAGTGGGTCGTCTTCGCGCTCAACCTGCTCAACACGAGCGCGATCCTGGGCGTCCCCGCGGCCGTCATCCACTACACACACTCTGAGCTCTTGCCCGGCTTTGCGCTCTGCACCGTCTCGGTCATCCTCTGGCTCAAGACCGTCTCCTATGCGCACTGCAACTGGGACCTGCGCATGGCGCACCGCGCGGGCGAGCTGCGCGACGGCGAGCGCGGCAGCGCCTGGGTCCCGCGCGACTGCGCCGCGCAGCTCAAGTACCCGGAGAACCTCACGCTCCGGAACTTGGGATATTTCCTCGCGGCCCCCACGCTGTGCTACCAGCTGAGCTACCCGCGGTCGGAGCGGTTCCGGCTCAAGTGGCTGCTGCGCCGCGTGCTCATGTTTGCCCTCACCATCACGCTGATGATGTTCATGATCGAGCAGTACGTGAACCCGCTCATCCACAACAGCCTGCAGCCGATGATGAGCATGGACTGGCTGCGCCTGTTCGAGCGCGTGCTCAAGCTGTCGCTGCCCAACCTCTACCTCTGGCTGCTCATGTTCTACGCGGGCTTCCACCTCTGGCTCAACATCGCCGCCGAGCTGACCATGTTTGGCGACCGCGAGTTCTACAAGGAGTGGTGGAACGCGACCACCATCGGCGAGTACTGGCGCCTCTGGAACCAGCCCGTGCACCAGTGGATGCTGCGCCACTGCTACTTCCCCTGCGTGCGCCACGGCGTGCCCAAGGTCTGGGCAGGCATCGTCGTCTTCTTCGTCAGTGCCGTCTTCCACGAGTGGATCGTCGCGCTGCCCCTGCACATGGTCAAGGGCTGGGCCPrototheca moriformis DGAT1-1, protein SEQ ID NO: 229MELCWPLLALLALADAAASEWVVFALNLLNTSAILGVPAAVIHYTHSELLPGFALCTVSVILWLKTVSYAHCNWDLRMAHRAGELRDGERGSAWVPRDCAAQLKYPENLTLRNLGYFLAAPTLCYQLSYPRSERFRLKWLLRRVLMFALTITLMMEMIEQYVNPLIHNSLQPMMSMDWLRLFERVLKLSLPNLYLWLLMFYAGFHLWLNIAAELTMFGDREFYKEWWNATTIGEYWRLWNQPVHQWMLRHCYFPCVRHGVPKVWAGIVVFFVSAVEHEWIVALPLHMVKGWAPrototheca moriformis DGAT1-2, nucleotide SEQ ID NO: 230CTGGAGCTGTGCTGGCCGCTGCTGGCGCTGCTGGCGCTGGCCGACGCCGCGGCCAGCGAGTGGGTCGTCTTCGCTTTGAACCTGGTCAACACGAGCGCGATCCTGGGCGTCCCCGCCGCGGTCATCCACTACACGCACTCTGAGCTGCTGCCCGGCTTTGCGCTCTGCACCGTGTCGGTCATCCTCTGGCTCAAGACCGTGTCCTACGCGCACTGCAACTGGGACCTGCGCATGGCGCACCGCGCGGGCGAGCTGCGCGACGGCGAGCGCGACAGCGCCTGGGTCCCGCGCGACTGCACCGCGCAGCTCAAGTACCCGGAGAACCTCACGCTGCGCAACCTGGGCTACTTTTTGGCGGCGCCCACGCTGTGCTACCAGCTGAGCTACCCGCGGTCGGAGCGGTTCCGGCTCAAGTGGCTGCTGCGCCGCGTGCTCATGTTTGCGCTCACCATCACGCTGATGATGTTCATGATCGAGCAGTACGTGAACCCGCTGATCCACAACAGCCTGCAGCCGATGATGAGCATGGACTGGCTGCGCCTCTTCGAGCGCGTGCTCAAGCTGTCGCTGCCCAACCTCTACCTCTGGCTGCTCATGTTCTACGCGGGCTTCCACCTCTGGCTCAACATCGCCGCCGAGCTGACCATGTTTGGCGACCGCGAGTTCTACAAGGAGTGGTGGAACGCGACCACCGTCGGCGAGTACTGGCGCCTCTGGAACCAGCCCGTGCACCAGTGGATGCTGCGACACTGCTACTTCCCCTGCGTGCGCCACGGCGTGCCCAAGGTCTGGGCCGGCATCGTCGTCTTCTTCGTCAGCGCCGTCTTCCACGAGTGGATCGTCGCGCTGCCCCTGCACATGGTCAAGGGCTGGGCCPrototheca moriformis DGAT1-2, protein SEQ ID NO: 231MELCWPLLALLALADAAASEWVVFALNLVNTSAILGVPAAVIHYTHSELLPGFALCTVSVILWLKTVSYAHCNWDLRMAHRAGELRDGERDSAWVPRDCTAQLKYPENLTLRNLGYFLAAPTLCYQLSYPRSERFRLKWLLRRVLMFALTITLMMEMIEQYVNPLIHNSLQPMMSMDWLRLFERVLKLSLPNLYLWLLMFYAGFHLWLNIAAELTMFGDREFYKEWWNATTVGEYWRLWNQPVHQWMLRHCYFPCVRHGVPKVWAGIVVFFVSAVEHEWIVALPLHMVKGWAPrototheca moriformis DGAT2, nucleotide SEQ ID NO: 232ATGCCCCGATTACGAAATATTGCTACTGCGTTGCTGATCGGGCCCGCTGCCCGTGCCTGCACCATGGGGCACGCCATTGACGTTCGACCCGTTGAACCGGACACACATTTTTTAAATTTAATTCATCTCAGTAGTCGTCGCAATGACGGAGGCCGTCTCGGACAGGGATGCTCTGCAACCGGAGCTGGGCTTCGTGCAGAAGCTGGGCATGTACTTTGCGCTGCCAGTTTGGATGATAGGCCTCGTGGTGGGCGCCCTCTGGCTTCCCGTGACCGTGGTGTGCCTCTTCATCTTCCCAAAGCTGGCGACATTGAGTCTGGGGCTGCTGCTGGTGGCGATGCTCACCCCCCTGTCGCTGCCCTGCCCCAAGCCGCTGGCCCGCTTCCTGGCCTACTGCACCACCGCCGCGGCCGAGTACTACCCCGTGCGCTTCATCTACGAGGACAAGGAGGAGATGGAGGCCACCAAGGGCCCCGTCATCATCGGGTACGAGCCCCACAGCGTCATGCCTCAGGCCATCTCCATGTTTGCCGAGTACCCGCACCCCGCCGTGGTCGGCCCGCTGCGCAAGGCGCGCGTGCTGGCATCCAGCACCGGCTTCTGGACGCCGGGCATGCGGCACCTGTGGTGGTGGCTGGGCACGCGCCCCGTGAGCAAGCCCTCCTTCCTCGCCCAGCTGCGCAAGCAGCGCTCCGTCGCCCTCTGCCCGGGCGGCGTGCAGGAGTGCCTCTACATGGCCCACGGCAAGGAGGTCGTCTACCTCCGCAAACGCTTTGGATTTGTCAAGTACGAGTTTGTGTGCAGCAAAGGGGGGTGGAGAGATGTCCTCCAATCTCACCAATCCAATCTACTCTTCCTCATGACTCCCGTTCATGTCACCGTACGCGCAGACTGGCCATCCAGACCGGCACCCCGCTGGTCCCGGTCTTCGCCTTTGGCCAGACGGAGACCTACACCTTTGTGCGGCCCTTCATCGACTGGGAGACGCGCCTGCTGCCGCGGTCCAAGTACTTTTCGCTGGTGCGCCGCATGGGCTACGTGCCCATGATCTTCTTCGGCCACCTGGGCACGGCCATGCCCAAGCGCGTGCCCATCCACATCGTCATCGGCCGGCCCATCGAGGTGCCGCAGCAGGACGAGCCCGACCCGGCCACGGTGCAGCAGTACCTCGACAAGTTCATCGACGCCATGCAGGCAATGTTTGAGAAGCACAAGGCCGACGCCGGCTACCCCAACCTGACGCTCGAGATCCACTGAPrototheca moriformis DGAT2, protein SEQ ID NO: 233MPRLRNIATALLIGPAARACTMGHAIDVRPVEPDTHELNLIHLSSRRNDGGRLGQGCSATGAGLRAEAGHVLCAASLDDRPRGGRPLASRDRGVPLHLPKAGDIESGAAAGGDAHPPVAALPQAAGPLPGLLHHRRGRVLPRALHLRGQGGDGGHQGPRHHRVRAPQRHASGHLHVCRVPAPRRGRPAAQGARAGIQHRLLDAGHAAPVVVAGHAPREQALLPRPAAQAALRRPLPGRRAGVPLHGPRQGGRLPPQTLWICQVRVCVQQRGVERCPPISPIQSTLPHDSRSCHRTRRLAIQTGTPLVPVFAFGQTETYTEVRPFIDWETRLLPRSKYFSLVRRMGYVPMIFFGHLGTAMPKRVPIHIVIGRPIEVPQQDEPDPATVQQYLDKFIDAMQAMFEKHKADAGYPNLTLEIH*Prototheca moriformis KASIII, nucleotide SEQ ID NO: 234ATGTCGACAGCTCGGGCAATGGCCGCGGGGCCGCCCTCTCGGTGTTTTGCTCCCAGCTCAACCCAGGCAGCCTGCGTCCGCCCGGTTAACCCCTCGCGAAGGGTCTGTGCCCGGGCGGGCATGCGCATGCTGGGCAGCGGTTCCTCCACCCCTTCCACGGTGCTGAGCAACGCCGATCTGGAGCAGCTGGTGGAGACGAACGACGAGTGGATCGTTGCCCGGACTGGCATCCGGCGCCGGCACATCCTGGGCCCTGGGGAGACGCTCACCCACCACTGCACCCTGGCCTGCCAGCGCGCGCTAGAGATGGCGGGCGTGGACGCCAAGGACGTGGACCTCATCCTGCTGGCCACGTCCAGCCCGGACGACTCCTTTGGCAGCGCCTGTGCTGTGCAGGCCGAGCTCGGGGCCAAGAGCGCGGCCGCCTACGACCTCACCGCCGCCTGCTCCGGCTTTGTCATGAGCACGGTCACCGCCACCCAGTTCTTGAGAACGGGGGTGTACAAGAACATCCTGGTCATCGGCGCGGACGCCCTGTCCCGCTACATCGACTGGCGGGACCGGAGCACCTGCATCCTGTTTGGCGACGGCGCCGGCGCCGTCCTCCTCCAGCGCGACGATCGCGAGGGCGAGGACTCCCTTCTCGGGTTCGACATGCACTCTGGTACGCTGCAATGGTCGAAGCGGGGCATCCATCCAACCCTTCCACACACCGNNNNNNNNNNCGCGGCCGCCTACGACCTCACCGCCGCCTGCTCCGGCTTTGTCATGAGCACGGTCACCGCCACCCAATTCTTGAGAACGGGGGTGTACAAGAACATCTTGGTCATCGGCGCGGACGCCCTGTCCCGCTACATCGACTGGCGGGACCGGAGCACGTGCATCCTGTTTGGCGACGGCGCCGGCGCTGTCCTCCTCCAGCGCGACGATCGCGAGGGTGAGGACTCCCTTCTCGGGTTCGACATGCACTCTGGTACGCTGCAATGGTGGGGGGTGGACGAAGAGGGGACGAATTGGAGCCATCAAGCTGCCCATGCGTGCTGTACTGTGCTCTTCCACGGGCCACACCTAGCATCCATCCAACCTTTCCACCCACGCTCTTCAGATGGGCACGGCCAGAAGCACCTCCACGCCGGCTTCATGGGCTGCGGCAACAAGGCCTTCTCGGAGCAGCCCTCCAGCGCCGCCGCTTTCGGCAACATGTACATGAACGGCAGCGAGGTCTTCAAGTTTGCCGTGCGAGCCGTGCCGCGGGTGGTGGAGGCCTCGCTGAAAAAGGCGGGGCTGAGCGTGTCCGACATCGACTGGCTGGTCCTGCACCAGGCCAACCAACGAATTTTGACGGCTGCCGCCGATCGGCTGGGTGTCCCCGCTGGTGCGCACGCATGCGGTGCTGCGGCTGGGGATGCGCTGTTCGACTGTGTGTCACAGCGCCATTTTGCCGACCCGGGCGCTCTGTGTGGTGGGATGCTCAACGCTCAGTCCCGATTTCTTAAGAAAGTGGTGTCTAATGTGGCCGAGTACGGCAACACCTCGGCCGCGTCCATCCCCCTCGTGCTGGACGAATCGGTCCGGCAGGGCCTGATCAAGCCGGGCGACATCCTGGGCATGGCAGGGTTTGGGGCTGGCCTGTCCTGGGCGGGAGCCATCCTCCGATGGGGC Prototheca moriformis KASIII, protein SEQ ID NO: 235MLGSGSSTPSTVLSNADLEQLVETNDEWIVARTGIRRRHILGPGETLTHHCTLACQRALEMAGVDAKDVDLILLATSSPDDSFGSACAVQAELGAKSAAAYDLTAACSGFVMSTVTATQFLRTGVYKNILVIGADALSRYIDWRDRSTCILFGDGAGAVLLQRDDREGEDSLLGEDMHSDELEPSSCPCVLCCALPWATPSIHPTLPHT????AAAYDLTAACSGFVMSTVTATQFLRTGVYKNILVIGADALSRYIDWRDRSTCILFGDGAGAVLLQRDDREGEDSLLGEDMHSDGHGQKHLHAGFMGCGNKAFSEQPSSAAAFGNMYMNGSEVEKFAVRAVPRVVEASLKKAGLSVSDIDWLVLHQANQRILTAAADRLGVPAEKVVSNVAEYGNTSAASIPLVLDESVRQGLIKPGDILGMAGFGAGLSWAGAILRWGPrototheca moriformis Choline Kinase, nucleotide SEQ ID NO: 236ATGGCGGGCGTCCCACACAGCTCTCTGGAGCTCGATCCGCTGTCGAGCAGAGCCGAGCTCGAGGTGGGGATCAGGGCGGTCTGCCGCGATCTGCTGCCGGGCTGGAAGGGCCTTGCCGACGATGCGATAGAGATCACCACCATTACGGGCGGGATCAGCAATGCGCTGTACAAGGCGACCCCGACCGTGGACGGGGACCTGGGACCGGTCCTCTGCCGGGTGTACGGCCTCAACACCGATCACTTTATCGAGCGGAAGAAGGAGGTGGCCATCATGCAGATTGTGAACGAGCACGGATTTGGCACCGAGGTGCTGGGGACGTTTGCGACGGGCCGGCTGGAGGCCTTTTTGCCTCAAAAGTGCCTGGAGCCGGAGCARCTCAAGGACCCCCAGATCTCRGCGGCCATTGCCCGGGCCATGGCCCGTTTCCACGCCGTCCCRGGGCCGGGCCCCAAGACGGCGTCCACGCCGTTTGGCCGCATCCACAAGTGGCTGGACATGGCGAGCGGGTTTACCTGGGACGACCCGCAGAAGCGGGAGGGGTTTGAGGCGTTTGACCTCTCGGAGCTGCGGCRGCAGGTGGAGGAGGTGGAGGCGCTCTGCRGCGCRGTCGGSGGCCCCATCGTCTTYGGGCACAACGATCTCCTGGCCGGCAACGTCATGGCCTCGGAGGCCTTTCTGCAGCGCGCGCAGCGCGGCGAGCGGCCGGACCCGGACKCGGACGTCCGCGCGAGCGCCGAGAGCGACCTGACCTTTATCGACTTTGAGTATGCCGACTGGACGCCGCGAGGATTCGACTGGGGCAACCACTTTAACGAGTGATTTGCGGGCTTCGACGGCAACTACGACAACTACCCAACCCCCGCCGAGGCGGCCGTGTTTGTGCGGGCCTACATGGCGGCGGAGCGCGGCTGCGAGGRRAGCGCGGTCCCGGACGCGGACGTGGACCGSGCGGTGRTCGAGGCGGAYCTGTTTGCGCTGGCYAGCCACCAKTACTGGGGCGCCTGGTGCTTCYTGCAGGCGCARTGGTCMAARCTKPrototheca moriformis Choline Kinase, protein SEQ ID NO: 237MAGVPHSSLELDPLSSRAELEVGIRAVCRDLLPGWKGLADDAIEITTITGGISNALYKATPTVDGDLGPVLCRVYGLNTDHFIERKKEVAIMQIVNEHGEGTEVLGTFATGRLEAFLPQKCLEPEQLKDPQISAAIARAMARFHAVPGPGPKTASTPFGRIHKWLDMASGFTWDDPQKREGFEAFDLSELR?QVEEVEALC?AVGGPIVEGHNDLLAGNVMASEAFLQRAQRGERPDPD?DVRASAESDLTFIDFEYADWTPRGEDWGNHENE*FAGEDGNYDNYPTPAEAAVEVRAYMAAERGCE?SAVPDADVDRAV?EADLFALASH?YWGAWCFLQAQWSKLPrototheca moriformis Pyruvate Dehydrogenase, Alpha subunit, nucleotideSEQ ID NO: 238ATGTCGTCAGTCGTGATCAGCTCCAGGACGGGCTGCCTCCCCCGCGCCGCGGCGGGGCCGGGCCGCCCCCAGCTCGCCTGCTCGTTCCGGCCGGGGTCCCACCCGCCCCGGGCGGGCGCCCCCGGCCCCCGCCCGCGCCGCGGCGTGGCCCCGCTGTCTGCCGTGGCTACCCCGCTCAAGGATGCGCCCGCGCAGCCCTCCAGCAAGCCCTCGATCAGCGCCGAGGTGGCCAAGGAGCTCTACCGGGACATGGTGCTTGGTCGGGAGTTTGAGGAGATGTGCGCGCAGATGTACTACCGCGGCAAGATGTTTGGATTCGTGCACCTCTACTCGGGCCAGGAGGCCGTGTCGACAGGCGTGATCAAGGGCTGCCTTCGCAAGGACGACTACATCTGCTCCACGTACCGGGACCACGTGCACGCGCTGAGCAAGGGCGTGGCGGCGCGCAAGGTGATGGCGGAGCTGTTTGGCAAGCGTACGGGCGTGTGCCGCGGCCAGGGTGGGTCCATGCACATGTTTGACGCGGAGCACGGGCTGCTGGGCGGGTACGCCTTCATCGGCGAGGGTATCCCCGTGGGCCTGGGCGCGGCCTTCTCGGTGGCCTACCGCCGCAACGTGCTCGGCGAGGACGGCGCGGACCAGGTGTCTGTCAACTTCTTCGGGGACGGCACCTGCAACGTGGGCCAGTTCTACGAGTCGCTCAACATGGCCAGCCTGTACAAGCTGCCCTGCATCTTTGTGGTGGAGAACAACCTGTGGGCCATCGGCATGAACCACCCGCGGTCCACGGGGCCCACGCTGGGCGACCAGGAGCCCTGGATCTACAAGAAGGGACCCGCCTTTGGCATGCCGGGCGTGCGCGTGGACGGCATGGACGTGCTCAAGGTGCGGGAGGTCGCGCTGGAGGCCGTGGAGCGCGCGCGCCGCGGCGAGGGGCCCACGCTGATCGAGGCCGAGACCTACCGCTTCCGCGGGCACTCCCTGGCCGACCCGGACGAGCTGCGCAGCAAGGAGGAGAAGGCCAAGTACGCGGCGCGCGACCCCATCCCGCAGCTCAAGCGCTTCATGCTCGACAAGGGACTGGCCACCGAGGAGGAGCTGCGCGACCTCGAGGCCGGCGTCGCCGCCGAGGTGGAGGAGTCGGTCACCTTTGCCGAGGCCAGCCCCAAGCCCGACATGTCCCAGCTCCTGGAAAACGTGTTTGCCGACCCCAAGGGCPrototheca moriformis Pyruvate Dehydrogenase, Alpha subunit, proteinSEQ ID NO: 239MSSVVISSRTGCLPRAAAGPGRPQLACSFRPGSHPPRAGAPGPRPRRGVAPLSAVATPLKDAPAQPSSKPSISAEVAKELYRDMVLGREFEEMCAQMYYRGKMFGFVHLYSGQEAVSTGVIKGCLRKDDYICSTYRDHVHALSKGVAARKVMAELFGKRTGVCRGQGGSMHMFDAEHGLLGGYAFIGEGIPVGLGAAFSVAYRRNVLGEDGADQVSVNEFGDGTCNVGQFYESLNMASLYKLPCIFVVENNLWAIGMNHPRSTGPTLGDQEPWIYKKGPAFGMPGVRVDGMDVLKVREVALEAVERARRGEGPTLIEAETYRFRGHSLADPDELRSKEEKAKYAARDPIPQLKREMLDKGLATEEELRDLEAGVAAEVEESVTFAEASPKPDMSQLLENVFADPKG*Prototheca moriformis Pyruvate Dehydrogenase, Beta subunit, nucleotideSEQ ID NO: 240ATGCAGAGCGCGCTCAAAACCGTGACCAACCCCGTGTCGGGGGTCTCGCCCCGCCCCGCCCCGGTCGGGTCCCGGGTGCCCGGCGTCCATGTGGCCCGGCGCCAGGTCTTCCGAGGTGGGCCCCTGCGTGCCAAGTCGGCCAAGAAGGAGATGATGATGTGGGAGGCCCTGCGCGAGGGCCTGGACGAGGAGATGGAGCGCGACCCCAAAGTCTGCCTCATCGGCGAGGACGTGGGTCACTACGGCGGTTCGTACAAGGTGTCGTACGGCCTGCACAAAAAGTACGGCGACATGCGCCTGCTGGACACGCCCATCTGCGAGAACGGCTTCCTGGGCATGGGCGTGGGCGCGGCGATGACGGGCCTGCGGCCCGTGGTGGAGGGCATGAACATGGGCTTCCTGCTGCTGGCCTTCAACCAGATCAGCAACAACTGCGGCATGCTGCACTACACGAGCGGCGGGCAGTTCAAGGTGCCCATGGTCGTGCGCGGGCCCGGCGGCGTGGGGCGCCAGCTGGGCGCCGAGCACTCGCAGCGGCTGGAGTCCTACTTCCAGTCCATCCCCGGCGTGCAGCTGGTGGCGGTGTCCACGCCCAAGAACGCCAAGGCCCTCTTCAAGGCCGCCATCCGCTCGGACAACCCCATCATCTTCTTCGAACACGTGCTCCTGTACAACATCAAGGGCGAGGTGGAGGGCCCCGACCACGTGCAGAGCCTGGAGCGCGCCGAGGTCGTGCGCCCGGGCGCGGACGTGACGGTGCTCTGCTACTCGCGCATGCGCTACGTGGCCATGCAGGCCGTGCAGCAGCTGGAGCGCGAGGGCTACGACCCCGAGGTCATCGACCTCATCTCCCTCAAGCCCTTCGACATGGAGACCATCGCGCGCTCCGTGCGCAAGACGCGGCGCGTCATCATCGTGGAGGAGTGCATGAAGACCGGCGGCATCGGCGCCTCGCTCTCCGCCGTCATCCACGAGTCCCTCTTCGACGACCTCGACCACGAGGTCATCCGCCTCTCCTCCCAGGACGTGCCCACCGCCTACGCCTACGAACTGGAGGCAGCAACCATCGTCCAGCCCGAAAAGGTCATCGCCGCCGTGAAAAAGGTCGTCGGCAGCCCCTCCAAGGCCCTCAGCTACGCGTGAPrototheca moriformis Pyruvate Dehydrogenase, Beta subunit, proteinSEQ ID NO: 241MQSALKTVTNPVSGVSPRPAPVGSRVPGVHVARRQVERGGPLRAKSAKKEMMMWEALREGLDEEMERDPKVCLIGEDVGHYGGSYKVSYGLHKKYGDMRLLDTPICENGFLGMGVGAAMTGLRPVVEGMNMGELLLAFNQISNNCGMLHYTSGGQFKVPMVVRGPGGVGRQLGAEHSQRLESYFQSIPGVQLVAVSTPKNAKALFKAAIRSDNPIIFFEHVLLYNIKGEVEGPDHVQSLERAEVVRPGADVTVLCYSRMRYVAMQAVQQLEREGYDPEVIDLISLKPFDMETIARSVRKTRRVIIVEECMKTGGIGASLSAVIHESLFDDLDHEVIRLSSQDVPTAYAYELEAATIVQPEKVIAAVKKVVGSPSKALSYA*Prototheca moriformis Pyruvate Dehydrogenase, DLAT E2 subunit, nucleotideSEQ ID NO: 242ATGCCGGCGCTCTCGCCCACGATGTCCCAGGGCAACCTGGTGGAGTGGACGGTGAAGGAGGGGCAGGAGGTCGCGCCCGGCGACTCCCTGGCCGAGGTCGAGACGGACAAGGCCACCATGACGTGGGAGAATCAGGATGACGGCTTTGTGGCCAAGATCCTGGTGCCCGCCGGCGCCCAGGGCATCGAGGTCGGCACGCCCGTCCTGGTACTGGTCGAGGACGAGGCGGACGTGGCCGCCTTCAAGGACTACAAGCCCCAGGGCCAGGCAGCGGCGGCCGGGGACAATAAGGAGGAGGGGTCCGAGAGCAAGGCCCCGGCCGCGAAGGCCGAGAAGGCGAGTGCGGCGTCTGGCGTGGCGAGGAACGACCACCTGCTTGGCCCCGCGGCCCGGCTGCTGCTGCAGGGCGTGGGGCTGTCGGTCGAGGACGTGACGCCCACGGGGCCGCACGGCGTCGTGACCAAGGGCGACGTCCTGGCGGCGATCGAGAGCGGCGCCAAGCCCGCGCCGAAGAAGGAGGAGGCGAAGAAGAGCGAGAAGGTGGTCGCGAAGAAGGAGGCGGCAACCCCCGCGCAGGAGGCCGCGGCGGCGCCGTCGGGCGGCCGCGCGCGGCGCCAGCGCGGCCAGTACACGGACGTGCCCAACTCGCAGATTCGGAAGATCATCGCCAGCCGCCTGTCCGAGTCCAAGTCGACCATCCCGCACCTCTACCTCTCCGCGGACGTGGACCTGACCGCGCTGGCGGCGCTGCGCGCCGCGCTCAAGGCGCAGGGCCTGAAGATCTCGGTCAACGACTGCGTGGTCAAGGCGGCCGCGGGCGCGCTGGCGGCCGTCCCCGCGGCCAACTTCCACTGGGACGACGCCGAGGAGCGGGCGCGGGCGGTGCGGAGCGAGGGCGTGGACATCTCCATCGCCGTGGCCACGGACAAGGGCCTCATCACGCCCGTCGTGCGCGGCGCCGACGGCAAGGCCTTAACCGCCGTGGCGTCCGAGATCCGGGAGCTGGCGGGCCGCGCGCGCAAGAACAAGCTGCGGCCGGAGGAGTTCCAGGGCGGCTCCTTCTCCATCAGCAATCTGGGCATGTTTGGCATCGACAGCTTCTGCGCCATCATCAACCCGCCCCAGGCCTGCATCATGGCCGTCGGCGCGGGGCGCGAGGAGACCGTGCTCGTGGACGGCCGGCCCCAGACCAAGACCATCATGACGGTCACCCTCTCCGCGGATCACAGGGTCTACGACGGTGAAATTGCGTCGGCGCTCCTGGAGTCCTTCAAGGCCAAGATCGAGGCGCCCTACACGCTCCTCCTGGCTTGAPrototheca moriformis Pyruvate Dehydrogenase, DLAT E2 subunit, proteinSEQ ID NO: 243MPALSPTMSQGNLVEWTVKEGQEVAPGDSLAEVETDKATMTWENQDDGFVAKILVPAGAQGIEVGTPVLVLVEDEADVAAFKDYKPQGQAAAAGDNKEEGSESKAPAAKAEKASAASGVARNDHLLGPAARLLLQGVGLSVEDVTPTGPHGVVTKGDVLAAIESGAKPAPKKEEAKKSEKVVAKKEAATPAQEAAAAPSGGRARRQRGQYTDVPNSQIRKIIASRLSESKSTIPHLYLSADVDLTALAALRAALKAQGLKISVNDCVVKAAAGALAAVPAANFHWDDAEERARAVRSEGVDISIAVATDKGLITPVVRGADGKALTAVASEIRELAGRARKNKLRPEEFQGGSFSISNLGMFGIDSFCAIINPPQACIMAVGAGREETVLVDGRPQTKTIMTVTLSADHRVYDGEIASALLESFKAKIEAPYTLLLA*Prototheca moriformis Acyl-CoA Binding Protein 1, nucleotideSEQ ID NO: 244ATGTCGAGCGACTTGGAGCCCAAGTTCAAAAAGGCCGTGTGGCTGATCCGCAACGGACCCCCGGCCGAGGGCGCCACCACCGAGACCAAGCTGCTCTACTACGCGCACTACAAGCAGGCCACGGAGGGCGATGTCACGGGGTCGCAGCCCTGGCGGGCGCAGTTCGAGGCCCGGGCCAAGTGGGACGCCTGGGCCAAGCTCAAGGGCATGAGYCGGGAGGAAGCTATGCAGAAATACATCGACCTCATCAGCAAGTCCAGACCCGACTGGGAGAACAACCCTGCTCTCAAGGACTACCAGGAGGAGTAG SEQ ID NO: 245Prototheca moriformis Acyl-CoA Binding Protein 1, proteinMSSDLEPKFKKAVWLIRNGPPAEGATTETKLLYYAHYKQATEGDVTGSQPWRAQFEARAKWDAWAKLKGMSREEAMQKYIDLISKSRPDWENNPALKDYQEE*Prototheca moriformis Acyl-CoA Binding Protein 2, nucleotide (transcript)SEQ ID NO: 246GCTCGGCCTCCTCCACCTCGCCCTCCTTCTCCTCCTTGGCCTCCTCATCACAAGTTCAATTTTTGATCACTTGCATGTATACGTTGTTTCGATACACGACATCGGCTCTGGGTCCCTCGAGAGTTCACTGATCCGTCCCACTTGCGGGCCGCACGGTGCGATGAGCGCCGCGCGCGAGGTGCAGGAGGAGGAGGAGGACCATTTCGAGGCGGCGGCCGAGCAACTGACCGTGACCATCGCGACGGGCACGAGACTGCCGGACGCCTCCCTGCTGGAGCTGTACGCCCTGTACAAGCAGGGCTCGGAGGGGCCCTGCTCCAGGCCCAAGCCGTCCTTCCTGGATCCCAAGGGCCGAACCAAATGGAAGGCCTGGCACGACCTGGGCGCCATGCCCCGCGATGAGGCCAGGGACAAGTACGTCGTCGCGGTGCGGCGACTGCTGGGCGGCGCGCCTGAGGGCCAGCCGCAGCCGGCGGCCTACGGCAGCACGGGCCCGGCGGTGAGCACCCCGGCCCGCGCCCTCGGCGAGGAGGCGGGGGACGCGGTGGGCGAGGGGGCCGACGACGTGGGGCCCCTCCACGCGGCGCTGGCCGATGGAGACGCGGCGCGGTTCGCGGCCGCCGTCGCCGAGGCCACGACCGGCCAGGCCGAGCGGCGGGACGCGGAGGGGGGCACCCCCCTCCACATCGCCGCGGATGCCGGCAACGTCGAGGCCATTGACCTCCTGAGAGCCAAGGGAGTCGACCTGGATCTGAAAGATCAGGACGGCATGACGGCGCTGCACTATGCGGCGCTGGCGAGCCAGCGAGAGGCGTACGAGGCCTTGCTTGAAGCGGGCGCGGATAGCAGTGTCCGAGACGCCGAGGGCCAGTCGCCCGCCGACCTGGCGCCCCCGGACTGGAACCGCGCGTGACGGGTGGGTTGTATGCCAAAACATTTGGAGACTGGAGTTTCCCAATCGGTGACTGGCCCCGCGCTTCGTGCCAAATTCTAGACAATTTCATGAGTCAGAGAGAGACGGCTCGGGTGCGGCTGCAACAGACTGCGCTTTCTGTAAGCAAGGCACTCTCGACAGATGGCGCTCCCGATGCCGGGCGCGAGCGCGCGCATCGCTACCCTGCGCGCTGCGCTTCTCCCTCCTCCTCGTGCAGGAGGCGGAGCCGCGCGGCGCGGATGACGTTGTGCATGGTGGCGGCGATGGTCATGGGCCCGATGCCGCCPrototheca moriformis Acyl-CoA Binding Protein 2, protein SEQ ID NO: 247LGLLHLALLLLLGLLITSSIFDHLHVYVVSIHDIGSGSLESSLIRPTCGPHGAMSAAREVQEEEEDHFEAAAEQLTVTIATGTRLPDASLLELYALYKQGSEGPCSRPKPSFLDPKGRTKWKAWHDLGAMPRDEARDKYVVAVRRLLGGAPEGQPQPAAYGSTGPAVSTPARALGEEAGDAVGEGADDVGPLHAALADGDAARFAAAVAEATTGQAERRDAEGGTPLHIAADAGNVEAIDLLRAKGVDLDLKDQDGMTALHYAALASQREAYEALLEAGADSSVRDAEGQSPADLAPPDWNRA* Prototheca moriformis Malic Enzyme, nucleotide SEQ ID NO: 248CGAGCCCTGGGGCCGTGCACCCTTTGGAGCAGCCAGGTGCAGCGACACCCTTTGGTACGCAGCCTGGCCACAGGCCCCTGCACTTTTACCTAGACTACATCTGATTGAATTTTATGTATGCTTGTCGTAATCGCGCATCTCCCGGTGTGCCCCATTGCCTGCAGTAGGCCTTCTCGATCGCTTACCCGGCTAGCAGCGTCTGGGTGCGGTGGATCGCCTCGGACGACCCCAACTTCCCCTACAAGCCCCTGGAGGTGAACGCCACGAGCGTGGTGCACCACCGCGGCATGGAGCTGCTGCAGAACCCGGTGTTCAACAAGGGCACCGCCTACTCTGTGGCCGAGCGGGAGCGCCTGGGCGTGCGCGGTCTGCTCCCGCCGCAGGTGCTGCCCGTGTCGCGCCAGATGGCGCGCGTCATGGACCGGTACTGGCACGGCTCCGACTTCATCAGCCCCGAGGAGATCGAGTCGGGCGGCATCACGCACGAGCACACCCGCAAGTGGCTCGTCCTGACCGAGCTCCAGGACCGGAATGAGACGCTGTTTTACCGCTGCCTGATAGAAAACTTTGTGGAGATGGCGCCGATTCTGTACACGCCAACGGTGGGCTGGGCGTGCCTGAACTACCACAAGCTCTTCCGCCGCCCGCGCGGCATGTACTTCTCGGCCTTTGACCACGGCAAGATGTCGACCATGGTGCACAACTGGCCGCACGAGGAGGTGGACGCGATCGTGGTCACGGACGGCTCGCGCATCCTGGGGCTGGGCGACCTCGGCTGCAACGGGCTGGGCATCCCCGTGGGCAAGCTGGACTTGTACGTGGCCGCGGGCGGCTTCCACCCCTCGCGCGTGCTGCCCTGCGTGGTCGACGTGGGCACCAACAACGAGGCCCTGCGCGCCGACCCGCAGTACCTGGGCGTCAAGCAGGCGCGCCTCGTGGGCGACGCCTACTACTCCCTGCTCGACGAGTTTGTGCAGGCCGTCATGCTGCGCTGGCCGCACGCCGTGCTCCAGTTCGAGGACTTTGCCATGGAGCACGCGGCGCCACTGCTGGCGCGCTACCGCAACCACCACACCGTCTTCAACGACGACATCCAGGGCACCGCCTGCGTCGCGCTGGCGGGGCTCTACGGCGCGCTGCGCGTGCTGGGCCGCGGGCCCGCCGACCTCACCGGCCTGCGCATCGCCGTTGTGGGCGCCGGCAGTGCCGGCATGGGCGTCGTGTCCATGATCGCGAAAGGCATGGAGCAGCACGGGCTGACGCCGGAGGAGGCGGCGGACAACTTTTGGGTGCTGGACGCCGACGGGCTGATTACGGAGGAGCGCGCCAACCTGCCGGCCCACGTCCGGCGCTTTGCGCGCTCCGACGAGCGCGGCACCGACGGCGAGCGCCTGCTGGAGACCATCCGCCGCGTCAAGCCGACCTGCCTCATCGGCCTCTCGGGCGCGGGGCGCCTCTTCATTAAGGACGTGCTCCAGGCCATGGCGGAGGTGACGCCGCGCCCCATCATCTTCCCCATGAGCAACCCCATCGCCAAGATGGAGTGCACGCACCAGGAGGCCATCAAGCACACCAAGGGCAAGGCGGTGTTTGCCTCTGGGAGCCCGCAGGGCCCGGTGGAGTGGGAGGGCCACTCGCACGCCCCCTCGCAGGCCAACAACATGTACATCTTCCCCGGCATCGCGCTGGGGGCCTGGCTGGCGCGGTCGGGCACGATCACGGACCGCATGCTCATGGCCTCGGCGGAGGCCCTGGCCCAGTGCACCACGCCCGCGGAGCTCGCCCTCGGCATGGTCTACCCCAGCATGACCCGCATCAGGGAAATTTCGCTGAGGGTGGCCGAAGCGGTGATCGAGGCCGCCGGCGCCTTGGCCCAGAACGAGCGCCTGCTCAAGGCAAAGGCCGAGGGGCCCGAGGCCCTGCGGGACTACATCCAGGCGCACATGTACCACCCAGAGTACACAACCTTGGTCTACAAAGAAAGCCGGTAAATTGCCGAGCTTGCTTGAACGTCTGATGTTTTGGCCCATAATGAGGCAGCCTCTGGCCAGCGCGTGCCCTTGCTTTCCCTTGCTGGTCCTTTTAAAGTTGGTTTCGCTCCCTATGGTGTGTTTGTGCGATGTGTAGAAAGTPrototheca moriformis Malic Enzyme, protein SEQ ID NO: 249LEVNATSVVHHRGMELLQNPVENKGTAYSVAERERLGVRGLLPPQVLPVSRQMARVMDRYWHGSDFISPEEIESGGITHEHTRKWLVLTELQDRNETLFYRCLIENFVEMAPILYTPTVGWACLNYHKLERRPRGMYFSAFDHGKMSTMVHNWPHEEVDAIVVTDGSRILGLGDLGCNGLGIPVGKLDLYVAAGGFHPSRVLPCVVDVGTNNEALRADPQYLGVKQARLVGDAYYSLLDEFVQAVMLRWPHAVLQFEDFAMEHAAPLLARYRNHHTVENDDIQGTACVALAGLYGALRVLGRGPADLTGLRIAVVGAGSAGMGVVSMIAKGMEQHGLTPEEAADNFWVLDADGLITEERANLPAHVRRFARSDERGTDGERLLETIRRVKPTCLIGLSGAGRLFIKDVLQAMAEVTPRPIIFPMSNPIAKMECTHQEAIKHTKGKAVFASGSPQGPVEWEGHSHAPSQANNMYIFPGIALGAWLARSGTITDRMLMASAEALAQCTTPAELALGMVYPSMTRIREISLRVAEAVIEAAGALAQNERLLKAKAEGPEALRDYIQAHMYHPEYTTLVYKESR*Prototheca moriformis ATP:Citrate Lyase Subunit A, nucleotide (transcript)SEQ ID NO: 250TTTTTTTTAGGGTATCCAGACTTCAACTGCTAGGTTGATCAGTGCATAATACAGTGGTGACCACAGCTGTCGAGGATGCTCTAGGGGGCACCGTATCCAGGCGTTCTAGCGAATCGACAATTGCACGAATAGAATCTATCATGCGCGATTGCGGGCAGGTGGGGGCGCATGAACAGCTTCGCCAGCCCTTGACGGGAGAAAAAGCTACATGCGCTTCTTGAGACGCGCATCTACAGACGCGCATCCAGTCCTGTACATCTTCAGATGCGCCTCGTGAGACGCGTGGATCCAGACACGCACCCCTGCCTTCAGGCGGCGCTGGAGGCCTTGGCGTCGGCGGCGTCGGCCTCGGCCAGGTGGTCGATGGCGCGCTTGCAGATGCTGGTCATGGTGGTCTCGGGGCCGTAGACCTCGATGTCGATGCCCGTCTCCTGGCCCAGCTTGCGCATGGCCTCCAGGCCGGCCCTGTAGTTGGGCCCGCCGCGGCGCACGAAGAGCTTGAGGCGCGCGCCGCGGATGGCGCCCTCGCGCTCGCGGATGGCGGCGATGATGCCGCGGAAGGTGGCGGCGACGTCGGTGAAGTTGGCGATGCCGCCGCCGATGAGCAGCGCGCGCGCGCGGCCGTCGGCGCCGGCGGTGGCGCAGTCGAGCAGGCGCAGCGCGTAGGCGTGCGTCTCGGCCGCGGAGGGCCCGCCCGAGTACTCGGCGTAGTTGCCCAGCTCCGCGGCCGCGCCGAGGTCGCCGACCGTGTCCGCGTAGATGACGCTGGCGCCGCCGCCGGCGACCATGGTCCACACGCGTCCGGACGGGTTGAGGATGCTCAGCTTCAGGCTCGCGCCCGTGCCCTCGTCCATGCCCGCCACGGCCGCCTCGGCCGCGCTCAGGCGGCGGCCAAAGGGCAGCGGGAACTCCAGGCCCTCGCCGCCCCACTTCTTGGCCGAGCGGAAGCCCGCGGTGTCGTCCAGCTCGCCGCGCATGTCCAGCGGGAAGGGGCGCCCGTCCGGCCCCAGCGTGAAGGGGTTCATCTCCATCAGCGTGAAGTCCAGGTCCACGTACACCGCGTAGGCCCCCGCGATGAAGGCCTCCAGCCCGGGCCGCACCTCCAGCGGCAGCGTGGCCACCAGCGGCGCCAGCTTCTCGCCGGACAGCTCCTCCTCCACGCCCACGCTGATCGTGCGCACAAGGTCCCAGTTGTCCTCGATCTCCATGCCGCCCGCCTCCGAGAAGGAGATGTCCGCGCCCGTGCGCGTGCCCTGGATGCAAAGGTAGTACTCCTCCTCGTGCGGCACAAACGGCTCGATGATGAAGCAGTCGATCACACCCTTGACCGTGCCCATGTCGATGACCTTGCCCATGCGCTGGCCGATAAACTCCTCAGCGCCGGCCAAGTCCAGCTTGAGGCCCACGAGGTCGTGCTTGCCGCGCTTTCCAAACAGACAATCGGGCTTGACCACCAGCGGGACCTGCGTCAACCAGGGGTGCTCGCCCACCAACTCGCCAAAGTTGGTGTCCTTTTTGACCTGGGCCACCCTGATGGGCAGGTCCAGGCCCGCCAGACGCGCCATGTGCGTCTTCAGCAGCGTCTTGGAGTTGTACTCCCTGATCTTTTTGCGTGCCATGGCGATCTAATGCGTCCCTGGTCTTGGTGGAGCCGGAGAGGCCTGGACAGAAAGCGATCGCGGGGTCGCTGGCGGGACGTCCGCGGACAACGAAGCGCGGCCCAAGCCGACCAGCACGCACCCGTCAAGCCTCACGGATGCCGAGCACGACGCGATTCGTGACTGTTGGAGCGGTAGGTAGCCTTGAATCCAGCAATACGGCCGTCTGGAACACTGAGCGGCTTGTGAGCGACCTCGCGCCTATGCAATAAGGGGTTGGTTTCGAGTGCTGACGAGGCTCTCCTAGCCTCGGGCACAGTGGGAATTCCTGGCCGCCAAAAAGGTCGCGAGCAGCCCCAGTGGGCGCTTTCAGTGATGTACAGAGCAGCCCGAGCGTTTATCTTACATTTTACTTGGTGACAGTGGATGGGTGTTTGAAATGAAGTTACCTGTGTCTGATCACTTATGATGACACCAGTGTTGGTTGGCACGGCGAATGCATATGGATGGCATGCAGAAGAATACACCCTCCGTTTTTGCTGCTGGCACAAATTCTCACTGCAAAGTCTCATGTTTATTCTGTGCAGTCCCTTCTTAGTTATAGACTCGGTCCAATTACGAACAATTTGCATGGGATGGGTGCTCAACTCATTCCCTGCCAGATACCACAAACAGACCCCAACATAGCCAGACTCATCTATTGTTGCAAGTCAAATCAAGCTGCTCACAAAAGTATGATTATGGTGGGAGCCTGTGCTTGCTCAAAGAATGTGATCACGAATGGCGCAGCCCTGCAGCATTTCCCGTGAATATATATGTATATAAACGAACACATCCAGGTATTGCCTGCAGCGTTATTCATTTTAGATTGATCAAGAAAGAGTAAGCTTTTGTGTGTTGTTGPrototheca moriformis ATP:Citrate Lyase Subunit A, proteinSEQ ID NO: 251MARKKIREYNSKTLLKTHMARLAGLDLPIRVAQVKKDTNEGELVGEHPWLTQVPLVVKPDCLEGKRGKHDLVGLKLDLAGAEEFIGQRMGKVIDMGTVKGVIDCFIIEPFVPHEEEYYLCIQGTRTGADISFSEAGGMEIEDNWDLVRTISVGVEEELSGEKLAPLVATLPLEVRPGLEAFIAGAYAVYVDLDFTLMEMNPFTLGPDGRPFPLDMRGELDDTAGFRSAKKWGGEGLEFPLPFGRRLSAAEAAVAGMDEGTGASLKLSILNPSGRVWTMVAGGGASVIYADTVGDLGAAAELGNYAEYSGGPSAAETHAYALRLLDCATAGADGRARALLIGGGIANFTDVAATERGIIAAIREREGAIRGARLKLFVRRGGPNYRAGLEAMRKLGQETGIDIEVYGPETTMTSICKRAIDHLAEADAADAKASSAA*Prototheca moriformis ATP:Citrate Lyase Subunit B, nucleotide (transcript)SEQ ID NO: 252GCCGGGGGCTGCGCCCTCATGCTTCCCGTGGCCCTCATTGTGAACAACCTCAGTCCTCACCGTCGCTACCCGACCTTCTGGTGGTGAGGGGCTGCAGCGCCTGTGTTATCTTACCGTGGTGGGTCCACATGTCTTGCGAGCAACAGTGGCCAGCGCAGACACGCGAGTGCCCACGTTTAGATATTTATGCCATCTTTCTTTGTTGAGTGAGCATCCACGCCAATAGCTCTCTTCAGGTGCAGGCTTTTCATTATATCATTTTGCGATTCGCATGCTGGTTCCTGTGTGCATTTACAATCCAGGTCGACAATTTCTTCCCACTGGCAAACCTGCACTCATCGCTACCCCACCAACCGTGGTTGCCTTTTTGCCTCCCATTGAGCCATTGCCAATATATAAAATATAATACCAACCCTACGTCATCATATCATTTATTCCATGTGCAAACCCTCGCTCGTACCAATGACAATGAGAAAACAAGGTGGCGTCCGCAGTGCTCAAGGCAAAGGGGGCGGCGGTTTGCCACGACCACCGGGGACGCCTTTCGAATGCCGCGTCAGCATGGTGAGCGGGAGGCATTAGCGAGGGATTGGCCAAAAGCGGTCGCTTCCGGCCTCCCAGCCCGTACCGGGCCCAAAGCCAATCGCCAAGGCCTCTACAGCCCACACGAAAGAGGTGCTACAGGCGCTCAGAGCGACCGCTCGATCACGATGCCCACGTCCCCACGCACGCCGGGCCGCTACGGCCAGGCGGGCCAGCTCTTCAGCAAGCAGACGCAGGCCATCTTCTACAACTGGAAGCAACTGCCAGTGCAGCGCATGCTGGACTTCGACTTTCTGTGCGGTCGCACCCTTCCCTCTGTGGCATGCATTGTCCAGCCCGGCAGCCAGCAAGGGTTCCAAAAGGTCTTCTTCGGCCCGGAGGAGGTGGCCATCCCGCAGTACGGCTCGATCGCCGAGGCGGTGGAGCAGCACCCGCGCGCGGACGTGCTGATCAACATGTCGAGTTTCCGGAGCGCGTTTGAGTCGTCGCTGGAGGCGCTGCAGCAGCCGACGATCCGCACGGTGGCCATCATCGCCGAGGGCGTGCCCGAGCGCGACACCAAGAAGCTGATCGCCGTGGCCCAGCGCGCGAACAAGGTCATCCTGGGCCCGGCCACCGTGGGCGGCGTGCAGGCGGGCGCCTTCAAGGTCGCGGACGCGGCCGGCACGCTGGACAACATCGTCGCCTGCAAGCTGTACCGCCCCGGCAGCGTGGGCTTTGTGAGCAAGTCGGGCGGCATGAGCAACGAGCTGTACAACGTGATCGCGCGCGCGGCGGACGGCATCTACGAGGGCATCGCCATCGGCGGCGACGCCTACCCGGGTTCGACGCTGTCGGACCACTGCCTGCGCTACCAGCACATCCCCGGCGTCAAGATGATCGTCGTGCTGGGCGAGATCGGCGGGCGCGACGAGTACTCGCTGGTCAAGGCGCTGGAGGCGGGCGCCATCACCAAGCCCGTCGTGGCCTGGGTGTCGGGCACGTGCGCGACGCTGTTCCAGACCGAGGTCCAGTTTGGGCACGCGGGCGCCAAGAGCGGCGGCGCCGACGAGTCCGCGCAGGCCAAGAACGCGGCGCTGCGCGCGGCGGGCGCGGTCGTGCCCGACTCGTTCGAGGACCTGGAGCCGACCATCAAGCGCGTCTACGACGAGCTGGTGGGCGCGGGCGCGATCGTGCCCGCGCCGCTGCCGCCGCCGCCCTCGGTGCCGGAGGACCTCGCGGCCGCCAAGCGCGCGGGGCGCGTGCGCGTGCCCACGCACATCGTCTCCTCCATCTGCGACGACCGCGGCGAGGAGCCGACCTTCAACGGAGAGTCCATGAGCGCGCTCATGGAGCGCGGCGCCACCGTCGCCGACGCCATCGGCCTGCTCTGGTTCAAGCGCCGCCTGCCGCCCTACGCCACGCGCTTCATCGAGATGTGCGTCGTGCTCTGCGCCGACCATGGTCCCTGCGTCTCCGGCGCACACAACACCATCGTCACCGCGCGCGCGGGGAAGGACCTCATCTCCTCCCTCGTCTCGGGCCTGCTCACCATCGGGCCGCGCTTTGGCGGCGCCATCGACGGCGCCGCGCAGCACTTCAAGGAGGCGGTTGAGAAAGGCTGGGAGCCCGACGAGTTCGTGGAGATCATGAAGCGCCGCGGCGTGCGCGTGCCGGGCATCGGCCACCGCATCAAGTCCAAGGACAACCGCGACAAGCGCGTGGAGCTGCTGCAGCGCTACGCCCGCGAACGCTTCCCGTCCACCCGCTTCCTGGACTACGCCATCGAGGTCGAGGGCTACACCCTGCAAAAGGCCGCCAACCTGGTGCTCAACGTCGACGGCTGCATCGGCGCGCTCTTCCTCGACCTCCTGCACTCGGTCGGCATGTTTGCCAAGCCTGAGATCGACGACATCGTCCAGATCGGCTACCTAAACGGCCTCTTCGTCCTCGCGCGCGCCATCGGGCTCATCGGGCACTGCCTCGACCAGAAGCGCCTGGGGCAGCCCCTCTACCGCCACCCCTGGGACGACGTCCTCTACACCPrototheca moriformis ATP:Citrate Lyase Subunit B, proteinSEQ ID NO: 253MPTSPRTPGRYGQAGQLFSKQTQAIFYNWKQLPVQRMLDFDFLCGRTLPSVACIVQPGSQQGFQKVFFGPEEVAIPQYGSIAEAVEQHPRADVLINMSSFRSAFESSLEALQQPTIRTVAIIAEGVPERDTKKLIAVAQRANKVILGPATVGGVQAGAFKVADAAGTLDNIVACKLYRPGSVGFVSKSGGMSNELYNVIARAADGIYEGIAIGGDAYPGSTLSDHCLRYQHIPGVKMIVVLGEIGGRDEYSLVKALEAGAITKPVVAWVSGTCATLFQTEVQFGHAGAKSGGADESAQAKNAALRAAGAVVPDSFEDLEPTIKRVYDELVGAGAIVPAPLPPPPSVPEDLAAAKRAGRVRVPTHIVSSICDDRGEEPTENGESMSALMERGATVADAIGLLWFKRRLPPYATRFIEMCVVLCADHGPCVSGAHNTIVTARAGKDLISSLVSGLLTIGPREGGAIDGAAQHFKEAVEKGWEPDEFVEIMKRRGVRVPGIGHRIKSKDNRDKRVELLQRYARERFPSTRELDYAIEVEGYTLQKAANLVLNVDGCIGALFLDLLHSVGMFAKPEIDDIVQIGYLNGLEVLARAIGLIGHCLDQKRLGQPLYRHPWDDVLYT Prototheca moriformis LEC2, nucleotide (transcript)SEQ ID NO: 254GAGTTTCAAGTTGTGCTCTCCACTTCCGCGCGCATTGCGACGAATTCTCACGCCCTTTGTGCAGGTGTCCAGTTCCACATCGGGAGGCATGGGGGGCCCGCCACCTCGGCGCGTCAAACGGCGTCAGGTAGGGCGGGCACGCTGGCACTCTTGCCCGATGACCCGTACCGAGCAGTACTGCCATGCCTCGGGTCGGAGACGGTCGGCACCGCCCCTGATCGCGACCACTTCCAAATGATCAAACCCTTGCAGACACGATGGACGTCGCGGCGGTGATACTGTCGCCCCAGACAACGCCGCGCCTGGCCGCAGGCCGGGTCGGTGTCGGACGCTCCGCGCAGGCGCCCAGACGACTGGGTGCCGGTGGCAGCGAGGACGAAGAAAGGGATGTACTGGACCTCAAGCTTCCAGGAGTGGGCGGGACGCCTCTGTCGCCCTGGAATGCGGTGATCCTGGAGGACCAGCCATCGACGAAGGACGACGCACGTGCCACGCAACACGATGAGAGTCCACGCCGACGGGCTTCCCGCCGCAAGATCTGAAGAGGCTCCGATTTAGAGTTCCATGGCCAATGGTTTTCTTGTACAAATGATTTACATGCAGTACTGTTCGAACACACCTCTCGACACCGGGCATATCGCCTCGCAGGGCAATCGAAGGCCGTCAAATCTGGCGGTAGTCGTGGGAAGTGACCCTGCCCGGGGGGGCAATCCGTACCATCAGCGAGGTGCGCAGGACCAGGCACGTCTGCCGTATATTTGAGGGCTTCCCTAGGGGTCGAGTCACCTTGCTCGGCTTCATTTTGCAGGTTTTACCAAAAAACCTGCTTGGCATTTTTCTTAACAGGGCATCTTGAGGTGCGACATACCTGATACCGACGAGCTCGAGCCAACCCTTCGCTCGCTGCGGCATTTTTGTAACGGTCGACCACGGAGCATTGCGGCAGCGATAATGTCCAATACTACGATCAAAGTACTGTGCTACAAGTCTCTGACGCACAGCGATGTGACGACGGAGATTGCCAAGTCTGGGCGACTGGTTCTGCCGAGGGCCCAGGTCACGGCCTGCCTGCAGCGCCTCCTGGCCCTCGGCAGCGCCGGCGGAGCCCCAGGCAAGGGTCGAGCGGGATCTCTGTGCGGTGCCGTCCCCTTGTTCATGCACGACATGGAGGGCAAGGTCTGGAACTTTCAGCTGAAGACATGGCACAACGTTGTGGCCTCTGGAGGCCGGGCAACCTACGTGTTGGAGAATACTGCTGGTTTTGTGCACGAAAACAAGCTTCAACCGGGAAGCTGGCTGGCTATTTGCGAGCAGGACGACCGCCTGGTCCTCCGCACCGACATCGTCAAGGGCCGCGATTTCCTGCTCCCCGTGGTGGCGCCCGCGACCACGGTCAAGAAGCCCTCCTCCCCCAGTTTTGCGCCCTGGTCCTCCAAGAGTTCGGACCTCTCGGCCGCCAGCATGACCCTGGTGAGCCTGGGCCACGACACGCCCTCCAGCAGCGAGGACAAGGCCTCCGCGCCGTCCCCCACCATCCTGTCGGTGCCGCTGGTGCGCCCGCGCCCCATGTACGGCGCCATCAGCGTCCAGCAGGTGCCGCGGTTGGGCTGGGCGCTGAGCATGGTGCCGCACCAGCCGCGCGGCCAGCCGTGCTGGTAACCCCGGGTCGCCCTTCCGGCGTGCTTCCAAGCACACCCCTGCCCCTTCCCAAGCCCGCGCGGCCCGCTCCGCGCGGCTTCCTCCCCCCGAACCCACCAACAACGTGACCTCGACGGCCACGCCCGTCGGGGCTCGGGACCTTGGCACCCCTTCAGTCGCACTACCCCGTCCCCACCCAATAACCAGTGTGCTTGGAAATAAACCGGCAGCGTCCTCAGCCGCCACCAGCTCACCCTCACCCCCGTTCCCTTACGCGCCCGCGGGACGCCCCTCGTCCCGGCAAGCCCGGGCGCACGTTGCCCACCCTCGATGTCTCACACGTGTACTCGCCCTTTTCTCGATCGGGCTCCCAAGCTTGTCAATTTTGCCGCGCGCGGCCCCTCTCTTGCACTGTGGCACCGGACCCAAACTCTCATTATTTTTGTTGCACTTGAACCCTGCACACCCGACCTTGGCCCCACCGGCACCTTGCGCAAGCCCAGAGTGTCCCGCAAATTCATTCTTGGTGCGGCTCGGCCGACTTCTGCCCAAAGCGCGCGGCCGCCCTCGTATAGATACTGCTCTTTTCTCTTACTGTGAGATCTCCCTCGGTACCCGGGGTCGCGGCTGCCAGGCGCCGCAGCAGGCCGCCAGGTGGCGTAGGCCGACCAGCGCCCAGAGCAGGTTGAGCAGGTAGAAAAAGTTGGCGTTCAGGCCGCCAAGATCCAGCCACACATGGGCCACAGCGGCGTGCGTCGCCAGCAGCGTCATCCACAGGCACTGCCAGGCGCTGGGGTCGCGCCCTCTCCGCCGCAGCACGGCCGCGCTCGTCGCCATGCACAGCGCCAGGCCA Prototheca moriformis Leafy cotyledon2 LEC2, proteinSEQ ID NO: 255MSNTTIKVLCYKSLTHSDVTTEIAKSGRLVLPRAQVTACLQRLLALGSAGGAPGKGRAGSLCGAVPLEMHDMEGKVWNFQLKTWHNVVASGGRATYVLENTAGFVHENKLQPGSWLAICEQDDRLVLRTDIVKGRDELLPVVAPATTVKKPSSPSFAPWSSKSSDLSAASMTLVSLGHDTPSSSEDKASAPSPTILSVPLVRPRPMYGAISVQQVPRLGWALSMVPHQPRGQPCW* Prototheca moriformis DGK/SpiK, nucleotides (transcript)SEQ ID NO: 256GGTTTGAGAGGAACAATTTGGGGATGGGCGGTGGCACTGGGATGAGGCACTGGGATGAGGCGCTCATTGTCAAAGAATAAAACGGAATGGACGAATCGGTCAGGGCCGCCAGCACCGGCCAGGGCCCAGGAGGCAGGCGTTTCAAGGGCGGGAATGGGGCCCCCAGCTAGTTTCGCGCTCGTCAGCCCTCCACGCCTCTGGCAAACACGTCGATCACTCCGCGGTGGAGCTGCGCGTTGATCGTGCGGCTTTGAATGAGCTCCCCATCGACGTTCCAGTGGCTCTCCTGGCCAACCGCTTCGATGTGCACCGCGTGGGCGGGCAGGACCTGGATGTAGGACCCGTGCTGCCCGCCCGCCTCGAGGCCGATGTGCGACATCCTGAGCAGAAACTTGAGGTACTGGAGGCGGCTGCACCGCCTGATGAGCACCAGGTGCAGCAGGCCGTCGCTCAGGTGACCATACTTGGCGACGCCCTTTTTCGACTTGTCAGAGCGGCAGGGCATGATGACGAGCATGATGCCTGCAAACTCCTGCTCCGGGAGGTGCACAAACTCTGCTCGCCGCCGCTCCCAGAGCTCCTCGGCCGAGGCGGTGAACCCCGCCGCGCCGTCGCGGCCCTCGCGCCCGCCGCGCGCGCAGAACTCGCAGTTGGCGAGGCAGGCGTGGCTGAAGCTGGCCCGACCCGGTCCGGCGGCCGGGGCCAGGTACGAGACGCGCGCGGCGTAGGCCCTGTTCGCGGCGAGCATCTTGGCGCCCACCAGCTCGTACCGCGCGGGCCCCAGCCAGCGCAGGCGCTCCGACTCGGCCATGAGGTCGCCCATGAAGCCGTAGCTGACCATGCAGGTCGCAAACTCGGGCAGGCCCGGCTCCCGCTCCAGACGCAGCACATCCAGCGCCACGGCGTCGCCCAGCACGACGTGCACCGCGGCCGTGAAGGCCGAACGCGTGCCGTTGAGCGTGCAGGCCACGGCGTCGGTGCTGCCGGCCGGGATGTGGCCGATGCGGAACTGCGCCCCCGGCGCGACGGCCGCGCACCCGGCCGCGCGCAGGCCCAGCACACCGTTGACGATCTCGTGGAAGAGCCCGTCCCCGCCCACGGCCACCACGCCGTCAAACGCGCGGCGCGAGGGTGAGCGCGAGCGCCGCGGCTTGTCGCGGTCGGCGCCAGCGGGGGACAGGGAACGGCGCTCCGCGCCCGCGTCAGCGTCGCTCTCGGCGCCGCCGCCGATCACCTGGCGCACGATGGTGACGGCGTGCTGCGGCTCCTGCGTCTCCACCACCGCGACCTTGATCCCGGCCTTTCGGAAGACGGGCGCCACGGTGGAGCTCCAGGTCGAGGGCGCCTGGCGCGAGCCGCCCACCGGGTTCACGATCACCAGCAGCCGCCGCGGGCGCCAGGTCTGCGCCCGCATGCCGGCTTGCAGCTGGGCGTGCCACGCACGTAGCAGCTCCTCGTCTGTGGAGACCAGGGTCACTTGGCGCGGATGCCAGTACGAGGGGTTGGAGGGCGCGCGCCGAAAGGTGAAGAGCGTGAGGGCCCAGGCCTTCTCGAGCCAGGAACACCACTGGCGACGGAGCGACGAGGGCTTGACCTCCGCCGCGATGATCTCTTGCAGCGGCAGCGACTCTGCACGCTGCCCGAAGCCTGGCAAGAGGTTGACGCAGCCGTCCATCTCAAACGCCCAGCTCAAGCCGCCCTCGCTGCCCGCCAGATCCAGACTGACTTGCGCCGGCACGCCGTCCACCTGCATCGGCACGCCCTGCAGCTCCGCCTCGCTGAGGCGCTTGGGAAAGGAGGAGGACCGCGATATCGAGGGGTGCATGAGTGGGGGCTGCCTGGGGTGCGCCATCAGGCTGGGCGACGCATTGGTACCTCGTGTCCGATGCGGAGAAGACAGCGATCCCGCCCGGGAAGAGGGCAGGCTCACGGAATCGTCGTCCATGTGCGATGACGGGCGCTGAAGCACCAGCTCGGTGCAGAGAACGTCACGGGGAGCTCCGCGCATGTCCCACTACCGACCCAACCGCACGCAGGGAGACGGTCCGTCAGGCATGAACGCGATGCGCATAAGGAAGCAACAACCATGCGTGTGTGGCAATCGAACGGCGAAATGGGCCGATTCTTGGCAAAGGGGGGAGGCTGGCGAGGGACGCACACCGCGTCTTCCGTCGGCATCACGTGGATCTGTCGTTCATGCATCGGGATGCCTTACATGCAACCATTGGCGCCGAGGCAGGCGCGAATGAGCCGAGCAACAGTGTACGCACGCCAGGAATCAATTATACTGACTAGACATTTCTGCCATGAACCTTTCGTACTCGCTCTCCACGGCAGCAGGCGCTGGCGGGGCGGGAGCGGGGGCGGGAGGGGGAGGGGGCGGCGGTGGCGCCTCGTCGTCGGCCATACCCCCGACTGGCGGGGGGGGGGGCGGCGGCGGAACGTCGTCCGTGGGCGGCGGTCCCGGGACCGCCGCCCCGCTAGCGTCGTACATGCCGTGGTACGCGCCCGCCTGATCGGGCGCGGGCGGCGGCACCGCCCCGTACTGCGCGCCGTAGGCGTACGGGTCGGCGGCGCCAGGGTAGGCCGCGTACGGGTCGGCTGGGGCCTCGGCGCCGGGGTACGCACCCGGCGGCGGGGGCACGGCGCCGTACGCCCCGCCGGGCGGCGGCGGCGGCGGGTAGGCGCCGCCGTAGGGCGGTGGTGGGGCGCCGTACCCCGGGCCGTA Prototheca moriformis DGK/SpiK, proteinSEQ ID NO: 257MQVDGVPAQVSLDLAGSEGGLSWAFEMDGCVNLLPGFGQRAESLPLQEIIAAEVKPSSLRRQWCSWLEKAWALTLFTERRAPSNPSYWHPRQVTLVSTDEELLRAWHAQLQAGMRAQTWRPRRLLVIVNPVGGSRQAPSTWSSTVAPVERKAGIKVAVVETQFPQHAVTIVRQVIGGGAESDADAGAERRSLSPAGADRDKPRRSRSPSRRAFDGVVAVGGDGLFHEIVNGVLGLRAAGCAAVAPGAQFRIGHIPAGSTDAVACTLNGTRSAFTAAVHVVLGDAVALDVLRLEREPGLPEFATCMVSYGFMGDLMAESERLRWLGPARYELVGAKMLAANRAYAARVSYLAPAAGPGRASFSHACLANCEFCARGGREGRDGAAGETASAEELWERRRAEFVHLPEQEFAGIMLVIMPCRSDKSKKGVAKYGHLSDGLLHLVLIRRCSRLQYLKELLRMSHIGLEAGGQHGSYIQVLPAHAVHIEAVGQESHWNVDGELIQSRTINAQLHRGVIDVFARGVEG*Prototheca moriformis Ketoacyl-ACP Reductase (KAR) nucleotideSEQ ID NO: 258TGGAGTTCAGACGTGTGCTCTTCCGATCTCGCGTGGTGAGCAAGCCTGCGCCGACGTGTTTGGTTTCTTCTTCATGTTGTTGCTCGGAGGCGCCGTCGATTAGTTTGCCACCTGACCACCGTGCCCACAGTCCCCGACCAGCTCACTGGCACTTTACTGCATGCTCATGCCGCCGTCGATGCTGAGGACCTGGCCGGTGATGTAGGCGGCGGCCGGGTCGGTGAGGAGGAACTTGACCAGCCCGGCGACCTGGTCGGGCGTGCCGTAGCTGTTGAGCGGGATCTTGGCGAGGATGGCCTTCTCGACCTCGGGGTTGAGCACGCCCGTCATCTCCGACTCGATGAAGCCCGGCGCGATGGCGTTGACGAGCACCTTGCGGCCGGCCAGCTCGCGCGCGAGGGAGCGCGTCATGCCGATGACGCCGGCCTTGGAGGAGGCGTAGTTGACCTGGCCCGCGTTGCCCATGAGCCCGACCACGGAGGTGATGTTGACGATGCGCCCGCGCCGCGCCTTGGCCATGACCTTGGCCGCGGCCTGGCTCGCGTAGAACACGCCGGACAGGTTGACGTCGATGACCTGCTGCCACTGCTCGGGCTTCATGCGCAGCAGCAGCGTGTCGCGCGTGATGCCCGCGTTGTTGACCAGCGCGTCCACGCGGCCCCACCGGTCCACCGCCGCCTTGATCAGCGCCTGCGCGTCCTCGATCTTGGACAGGTCCCCCTTGACCACGATCGCCTCGCCGCCAGAGGCCTCGATCTGGTGCGCCACCTCCTCCGCGGCCCCGCTGGACGCCGCATAGTTGACCAGCACCTTGCCGCCCGCGGCGCCCAGCTTGAGCGCGATCGCGCGGCCAATGCCGCGCGAGGAGCCCGTCACCACCGTCACGCCCTGCTCCAGCTCCTTCTGGAGGGAGACGCCGTTGGGCGACGAGGCCACGGAGGCAGCGATCCATGCCGATCAACAGCCGGCTTTTCAGAACCTTAATCATGCAGAGCTCCGTGATGAACGTGCCGGCGACCCTTTCGGCGGGATGTGCCCTTAGGGCCTCCCCCCGCCTGCCGGCCGCTCGCCCGGCGCAGCACGCTGCCGGTCGCCGGTCGCCGGGCCAGCTGCAGGTCACGCGCGCCGCTGCCTCCGTGGCCTCGTCGCCCAACGGCGTCTCCCTCCAGAAGGAGCTGGAGCAGGGCGTGACGGTGGTGACGGGCTCCTCGCGCGGCATTGGCCGCGCGATCGCGCTCAAGCTGGGCGCCGCGGGCGGCAAGGTGCTGGTCAACTATGCGGCGTCCAGCGGGGCCGCGGAGGAGGTGGCGCACCAGATCGAGGCCTCTGGCGGCGAGGCGATCGTGGTCAAGGGGGACCTGTCCAAGATCGAGGACGCGCAGGCGCTGATCAAGGCGGCGGTGGACCGGTGGGGCCGCGTGGACGCGCTGGTCAACAACGCGGGCATCACGCGCGACACGCTGCTGCTGCGCATGAAGCCCGAGCAGTGGCAGCAGGTCATCGACGTCAACCTGTCCGGCGTGTTCTACGCGAGCCAGGCCGCGGCCAAGGTCATGGCCAAGGCGCGGCGCGGGCGCATCGTCAACATCACCTCCGTGGTCGGGCTCATGGGCAACGCGGGCCAGGTCAACTACGCCTCCTCCAAGGCCGGCGTCATCGGCATGACGCGCTCCCTCGCGCGCGAGCTGGCCGGCCGCAAGGTGCTCGTCAACGCCATCGCGCCGGGCTTCATCGAGTCGGAGATGACGGGCGTGCTCAACCCCGAGGTCGAGAAGGCCATCCTCGCCAAGATCCCGCTCAACAGCTACGGCACGCCCGACCAGGTCGCCGGGCTGGTCAAGTTCCTCCTCACCGACCCGGCCGCCGCCTACATCACCGGCCAGGTCCTCAGCATCGACGGCGGCATGAGCATGCAGTAAAGTGCCAGTGAGCTGGTCGGGGACTGTGGGCACGGTGGTCAGGTGGCAAACTAATCGACGGCGCCTCCGAGCAACAACATGAAGAAGAAACCAAACACGTCGGCGCAGGCTTGCTCACCACGCGAGATCGGAAGAGCACACGTCTGAACTCCAPrototheca moriformis Ketoacyl-ACP Reductase (KAR) proteinSEQ ID NO: 259MQSSVMNVPATLSAGCALRASPRLPAARPAQHAAGRRSPGQLQVTRAAASVASSPNGVSLQKELEQGVTVVTGSSRGIGRAIALKLGAAGGKVLVNYAASSGAAFEVAHQIEASGGEAIVVKGDLSKIEDAQALIKAAVDRWGRVDALVNNAGITRDTLLLRMKPEQWQQVIDVNLSGVFYASQAAAKVMAKARRGRIVNITSVVGLMGNAGQVNYASSKAGVIGMTRSLARELAGRKVLVNAIAPGFIESEMTGVLNPEVEKAILAKIPLNSYGTPDQVAGLVKFLLTDPAAAYITGQVLSIDGGMSMQ* Prototheca moriformis Lipoate Synthase (LS) transcriptSEQ ID NO: 260ACTCAATCGACCCGCACGCGAGTACGAAAAGATTCCATGGTGCGAAACGTTGAATGCCATAGTTTGTAACGGGCCAGGCGTACGCGTGCGGGTCGATTGAGTTGACGTCCCGTAGCCCCGCTGGGCGGTCACCTGAGTGAATTTGGAGCGAGCCGCATCCGACGACGGGTACCTGTCATTCGAAACACCCGTATCATTACGGGAGTTTTGTATTGCCAAACTTTTATAATTGAACTGCCCGACAGTACATTCCGCTTGGAAAGCATGTCGTCTCGGATCGACATGGGCGTGTCGACGTCCGGGAGGGTGGGCAACCCAATGACTGAGCGATCAGCCGCCCACCAATTGCGGGCGACCGCTGCACGCGTGCCAAGACGGCATGCACCCTTCAAATCCGCCGCAAGGCATCGCGGGCCAGCGGCGATGCGCTCGGCGGCGTTTGACGAGCTGGTGGCGCTGGCCCGGGCCAAGGACCCCACCCTGCCCCTGCCCAAGCGCAAGCCGGCCAGCCCGCGCCCCAAGCCCCCCAAGGCGCCCTGGCTGCGGCAGCGCGCCCCCCAGGGCGAGCGCTACGAGTACCTCAAGCAGCAGCTGACGGGCCTGAAGCTGGCGACCGTGTGCCAGGAGGCGCAGTGCCCCAACATCGGCGAGTGCTGGTCGGGCGGCGCCGAGGGGATCGGCACGGCCACCATCATGGTGCTGGGTGACACCTGCACGCGCGGCTGCCGCTTCTGCGCTGTCAAGACGTCGCGTGCGCCGCCGCCGCCGGACGCGGACGAGCCCGAGAACACGGCCGCGGCGGTGGCCGACTGGGGCGTCGGGTACGTGGTGCTGACCTCGGTCGACCGCGACGACCTGCCCGACGGCGGCGCGGGTCACTTTGCGGCCACGGTGCGCGCGATCAAGCGCCGGCGCCCCGAGGTCCTGGTCGAGTGCCTGACCCCCGACTTTTCGGGGGACGAGGCGCTCGTCGCCGAGCTGGCGCGGTGCGGGCTAGACGTCTTTGCGCACAACGTGGAGACGGTGGCGCGCCTGCAGCGGCGCGTGCGTGACCACCGCGCCGGCTACGAGCAGACGCTGGCCGTGCTGCGCGCGGCCAAGCGCGCCTGCCCCTCGCTGCTCACCAAGTCCTCCATCATGCTCGGGCTCGGCGAGCGCGACGAGGAGGTCGAGCAGACCATGCGCGACCTGCGGGGGGCGGGCGTGGACATCCTGACCCTGGGGCAGTACCTGCAGCCCACGCACCGCCACCTGGAGGTCCAGGACATGGTCCACCCAGACGCCTTTGACCACTGGCGAGAGGTCGGAGAAGCAATGGGGTTCCGGTACGTGGCCTCGGGTCCGCTGGTGCGGTCCTCGTACAGAGCCGGCGAGTTTTACGTGGCAGCCATGCTCAGGGAAGGAAAGCGCGGCGGCGCGGGGGCGCAGTTGTCGCCCTGAGGCGGCGGTGGCAGTCTCTTGAGGCTTTTGGGTGCGCAATCTCAAACACGTGTGTCTGTCCTGCGCGGGACCGAGAGGCATCATATTCCGCACGAGCGTGCATCATCCGGATGCAAACACCTATTCTCGTATTCTAATCTTGTGAATTATAGGCTGGCGTCACCAGCTGCTGAATCATGCGTTTTGGAGGCACCGCCCCTGAATATTTGGCGGCCCACTGCAACGCTGTAAGAATCTTCTTGCCCTTTCACCCGGAAGTTCAAATATCTCTGCGTAAACAGCAGGCGTAGAAACAGAAACGAGCGACGCATGGTAGCGGGAGGAACGAAACAAGGCGCGCTGCTCAGGGGCAAGGGGCATTCGTCATCACAGATTCCGCCCAGCTCCCAATTTCCCCAACCACTGCTAGATCTGGCGCGCGCCGCGGCTGTCGAGGCGGACCACCTGACTGGAGTTGGTCTCCAGCCCGCCGTGGCGGAGAAAGGCCTCGGACATGGCGCGCGCGACGCGCTCGCCGGTGGCCGGGTCCGGCACGACCGCCACGGCCPrototheca moriformis Lipoate Synthase (LS) protein SEQ ID NO: 261MSSRIDMGVSTSGRVGNPMTERSAAHQLRATAARVPRRHAPFKSAARHRGPAAMRSAAFDELVALARAKDPTLPLPKRKPASPRPKPPKAPWLRQRAPQGERYEYLKQQLTGLKLATVCQEAQCPNIGECWSGGAEGIGTATIMVLGDTCTRGCRECAVKTSRAPPPPDADEPENTAAAVADWGVGYVVLTSVDRDDLPDGGAGHFAATVRAIKRRRPEVLVECLTPDFSGDEALVAELARCGLDVFAHNVETVARLQRRVRDHRAGYEQTLAVLRAAKRACPSLLTKSSIMLGLGERDEEVEQTMRDLRGAGVDILTLGQYLQPTHRHLEVQDMVHPDAFDHWREVGEAMGFRYVASGPLVRSSYRAGEFYVAAMLREGKRGGAGAQLSP*Prototheca moriformis Homomeric Acetyl-CoA Carboxylase, NucleotideSEQ ID NO: 262ATGACGGTGGCCAATCCCCCGGAAGCCCCGTTCGACAGCGAGGGTTCCTCGCTGGCGCCCGACAATGGGTCCAGCAAGCCCACCAAGCTGAGCTCCACCCGGTCCTTGCTGTCCATCTCCTACCGGGAGCTCTCGCGTTCCAACCAGCAGTCGCGGTCGTTCACTTCGCGGGGGGTGCCAGGGAGGACGGACGTTTCGGATGAGCTGGAGCGCCGCATCCTCGAGTGGCAGGGCGATCGCGCCATCCACAGCGTGCTGGTGGCCAACAACGGTCTGGCGGCGGTCAAGTTCATCCGGTCGATCCGGTCGTGGTCGTACAAGACGTTTGGGAACGAGCGTGCGGTGAAGCTGATCGCGATGGCGACGCCCGAGGACATGCGCGCGGACGCGGAGCACATCCGCATGGCGGACCAGTTTGTGGAGGTCCCCGGCGGCAAGAACGTGCAGAACTACGCCAACGTGGGCCTGATCACCTCGGTGGCGGTGCGCACCGGGGTGGACGCGGTCGACTGCGGGGGGCAGATCCGGTTCGTGCGCACCGGGGTGGACGCGGTGTGGCCCGGCTGGGGCCACGCGTCCGAGTTCCCGGAGCTGCCCGAGTCGCTGGGCGCCACGCCCTCGCAGATCCGGTTCGTGGGCCCGCCCGCGGGGCCCATGGCGGCCCTGGGCGACAAGGTGGGCTCCACCATCCTGGCGCAGGCGGCGGGCGTGCCCACGCTGGCCTGGTCCGGCTCGGGCGTGAGCATCGCCTACGCGGACTGCCCGCGCGGCGAGATCCCGCCCGAGGTCTACCGCCGCGCCTGCATCGACTCGCTGGAGGCGGCGCTGGCCTGCTGCGAGCGCATCGGCTACCCGGTCATGCTCAAGGCCAGCTGGGGCGGCGGCGGCAAGGGCATCCGCAAGGTGCTCAGCGCCGACGAGGTCAAGCTGGCCTACACCCAGGTCTGCGGCGAGGTCCCCGGCTCGCCCGTGTTCGCCATGAAGCTCGCGCCGCAGTCGCGCCACCTGGAGGTCCAGCTGCTCTGTGACGCGCACGGCCAGGTCTGCTCGCTCTACTCGCGCGACTGCTCCGTGCAGCGCCGCCACCAGAAGGTGGTGGAGGAGGGGCCCGTCACCGCCGCGCCGCCCGAGGTGCTGGAGGGCATGGAGCGCTGCGCGCGCTCCCTGGCGCGCGCCGTCGGCTACGTCGGCGCCGCCACGGTCGAGTACCTCTACATGGTCGAGACGCGGGAGTACTGCTTCCTGGAGCTCAACCCGCGCCTGCAGGTGGAGCACCCGGTGACGGAGATGATCACGGGCGTGAACATCCCCGCCGCGCAGCTGCTCGTCGCGGCCGGCGTGCCCCTGCACCGCATCCCCGACATCCGCAGGCTGTACCGCGCGCCGCTGGCCGAGGACGGCCCCATCGACTTTGAGGACGACGCCGCGCGCCTGCCGCCCAACGGGCACGTGCTGGCGGTGCGCGTGACGGCCGAGAACGCGCACGACGGCTTCAAGCCCACCTCGGGCGCGATCGAGGAGATCAGCTTCCGCTCCACGCCCGACGTCTGGGGCTACTTCTCGGTCAAGGGCGGCAGCGCCGTGCACGAGTTCGCCGACAGCCAGTTCGGCCACCTGTTCGCGCGCGGCGACTCGCGCGAGGCGGCCGTGCGGGCCATGGTGGTGGCGCTCAAGGAGATCAAGATCAGGGGCGAGATCCAGACGCCCGTCGACTACGTCGCGCGCATGATCCAGACCGACGACTTCCTGGGCAACCGCCACCACACGGGCTGGCTGGACGCGCGCATCGCGGCGCAGGTCGGCGCCGAGCGCCCGCCCTGGTTCCTGGTCGTCATCGCGGGCGCCGTGCTGCGCGCGACGCGCGCCGTGAACGAGCGCGCGGCCGCCTTTTTGGACCACCTACGCAAGGGCCAGCTCCCGCCCAGCGAGGTCCCGCTCACGCGCGTGGCCGAGGAGTTTGTGGTCGACGGCACCAAGTACGCGGTCGACGTCACGCGCACGGGGCCGCAGGCCTACCGCGTCGCGCTGCGCGACCCCGAGGGCGACGGCGCCCCCGGGCACGCGCGCCGCGAGAGCCTCGCCTCCGCGGCCGGCGCGGCCAGCTCCGTCGACGTCATCGCGCGCACGCTGCAGGACGGCGGCCTGCTGCTGCAGGTGCGACGGGCGCGTGCACGTGCTGCACAGCGAGGAGGAGGCGCTGGGCACGCGCCTGGTCATCGACTCGGCCACGTGCCTGCTCAGCAACGAGCACGACCCCTCGCAGCTCGTGGCCGTCAGCACGGGCAAGCTCGTGCGCCACCTCGTCGCCGAGGGCGAGCACGTGCGCGCGGACGAGCCCTACGCCGAGGTCGAGGTCATGAAGATGATGATGACGCTGCTCGCGCCCCGCGGCCGGCGCGGTGCGCTGGGAGGTGGCCGAGGGCGCCGCGCTGGCGCCGGGCCTGCTGCTCGCGCGCCTGGAGGAAGCTGGGACGACCCTCCCGCCGTGCGCCCGCGAGCCCTTCCGCGGCGCCTTCCCCGACCTGGGCCCGCCCTCGCCCGACTTTGACGGCCTGGCCGCGCGCTACCGCTCCGCGCTCGAGGCCGCGCAGGACGTGCTCGACGGCTTCGAGGGCGACGTGCCCGCGGTCGTCGCCGCGCTGCTGGCCGCGCTGGACGACCCCGCGCTCTCCCTCGTCCTGCTGGACGAGGCGCTGGCCGTGGTCGCGCCGCGGCTGCCGGCCGCGCTGGCCGCGCGCCTGGAGGCCGCGGCTGGCCGAGGGCGCCGCCGAGATGGCGGGCGAGCTCTCGGACGACGAGCAGGACGCCGCGCTGGCGGCGCCGGGTCGCCGCGCGAAGGGAGGGGCCTCCACGCGCGCTTCGTGGTCAAGCGCCGCGACCCGGCGGCGCGCCTGCAGCCGCGCGTCTTCGAGGAGGGCCGCCGGCGCGCGGCGCTGGCCTCGCCGCCTCCTGCTCGAGCGCTACCTGGACGTGGAGGAGCGCTTCGAGTCCGGCGGCGCCAAGACGGACCAGGAGGTCATCGACGGCCTGCGCGGCACGCACAGCGCCGACCCGGGCAAGGTGCTGGAGATCGTGGTCGCGCACCAGTCGGCCGGCCCGCGCGCCGACCTGGTGCACCGCCTGCTCAACGCGCTGGTGCTGCCCTCGCCCGAGGCCTACCGCCCCATCCTGCGCCGCCTGGCCGCGCTGACGGGCGCCGGCAGCGCCGCGCCCGCGCAGCGCGCGCGCCGCCTGCTGGAGCACAGCCTGCTGGGCGAGCTGCGCGTGCTGGTCGCTGCGCGCGCNGTCCGGCCNTGGACATGGTTCAGCGACGCCGCGCTGCGCGGGCTGTGCCTGGGCGAGTCGCCGGGCGAGCCCGTCTCGCCGCGCGCGGAGCTGGCCAGCGCGCTCGCGGCCGAGTCCTCGGTCGCGTCGCCCACCTCGCGGCGGTCCACGCTCGCGGAGGGGCTCTACGTCGGCCTGGGCAACCTGGCGGCTGCCGCCGCGAGCAGCGTGGAGGCGCGCATGGCCATGCTGGTCGAGGCCCCCGCCGCGGTCGACGACGCGCTGGCCACGCTGCTGGACCACCCGGACCCGGTGCTCCAGCGCCGCGCGCTGTCCACCTACGTCCGCCGCATCTACTTCCCGGGCGTGCTGCACGAGCCGCAGGTCGTCGGGCGTCGGGCCGGCGCGCGGTCCGTCCGCGTCTGGGCGACGCGGGGCGCGCCCGGCGCGATGGCGCCGCGGCCCAGCAGGACCAGCATGGCCGGGCTCGCGCCGGGGACGCTGCACGTGGTCCTGACCGCCGAGGGCGCCGCCGCGCTGCAGCTGGACGCCGCCGCGCAGGCCGCGCTGGGCACGCTTGACGTCTCCGGCTACGTCGCGCCCGAGCACCAGCAGCAGCCGGGGCTGCCGGACGCCTCGGTCGTCGCGGCCAGTGCCGCCGCCGCGGTGTCGGCGCTCGCGCCCGCGCTGCGCGAGGCCGGCTACAGCGCCGTCTCCTTCCTCACCAAGCGCGGCGGCGTCGAGCCCCTGCGCGTCGTGTTTTACGACGGCACGTCGAGCGGCAGCGCTGCCGCGCCCGACTCCTCGTCCACCCAAACTGGCCATCGACCGGCTGGTCCCTGGACCCCGTGCTCGGCACGGTCGAGCCGCCGACGGCCGAGGCGCTGGAGCTGGCCTGTCTGGCCGGCCGCGCGGGCGCCTCGCACGACGCCTCGCGCAACCGTGGCACATGTGGACCGTGGCCGAGCGCGCCGGCAAGCGCAGCGCGGTGCTGCGCCGCACCTTTTTGCGCGGCGCGGTGCGCTCGCTGGGCCGGCCGGCGCTGCTGAGCGCGGCCTACGCGGGAACGGCCCCGCTGTCGCGGCGGCGGCGCTGGCCGAGCTGGAGCAGACGGTGGAGGCGGCGGCCGCCGAGCTGGAGCGGCTGGGCAAGGGCCGCGTGGGCGGCGCCTCGTCCCGACTGGACGCACCGTGCTTGTCGGTGCTGATGCTCGCTGCCGCTGGGCGCGTCCGAGCCGCGCGAAGGGAAAGGCGCCGTCGCGCGCGCGCTGGCCGCGCCGCGGCCATCGCCTCGCGCCACGTGGCGGCCCTGCGCCGCGCGGCGCTGGCGCAGTGGGAGGTCTGCCTGCGCACCGGCTCGGGCGGCGCCTCGCACCGCGAGGGCGGCTGGCGCGTGGTCGTGTCCTCGCCCACGGGGCACGAGGCCGGCGAGGCCTTTGTGGACGTCTACCGCGAGGCCGCCGACGGCACGCTGCGCGCGGTCAACCCCAGCCTGCGCGCGCCGGGCCCGCTGGACGGCCAGTCCGTGCTCGCGCCCTACCCGACGCTCGCGCCGCTGCAGCAGCGCCGGCTGACGGCTCGGCGGCACAAGACCACGTACGCGTACGACTTCCCGGCCGTATTCGAGGACGCGCTGCGCTCGATCTGGCTGCAGCGCGCCGTGGAGCTGGGCGCCACCGGGCTGGAGGCCGCGCGCGAGGACCTGCTGCCGCCCGGGACCGCCTGGTCACGGCCGGAGCTGGTGCTGGACACGGAGGAGGCGATCTACGAAAACGGCGCCGCGCACATTCGCCTGACCGACCGCGCGCCCTCGATGAACGACCTCGGCGTCGTGGCCTGGCGCCTGACGCTGGCCACGCCCGAGTGCCCGCGCGGCCGCGCCGTCGTGGCCATCGCCAACGACATCACCTACAACTCCGGCTCCTTCGGCCCGCGCGAGGACGCCTTCTTCAAGGCCGCCACCGAGTACGCGCTGGCCGAGCGCCTGCCGGTGGTCTACCTGGCGGCCAACTCCGGCGCGCGCGTCGGGCTGGCCGAGGAGGTCAAGCGCTGCCTGCGCGTGGAGTGGAGCGTGCCGGGCGACCCGACCAAGGGCCACAAGTACCTCTACCTGGACGACGAGGACTACCGCTCCATCACGGGCCGCGCCGCGGGCCGCACGCTGCCCGTCAGCTGCTCGGCCAAGGTCGGCGCCGACGGGCGCACGCGGCACGTGCTGCACGACGTCATCGGGCTCGAGGACGGGCTGGGCGTCGAGTGCCTGAGCGGGTCCGGGGCCATCGCCGGCGCCTTTGCGCGCGCCTTCCGCGAGGGCTTCACCGTCACGCTCGTGTCGGGGCGCACCGTGGGCATCGGCGCCTACCTCGCGCGCCTGGGCCGGCGCTGCGTGCAGCGCCGCGAGCAGCCCATCATCCTCACCGGCTTCGCCGCGCTCAACAAGCTGCTGGGGCGCGAGGTGTACACCTCRCAGCAGCAGCTGGGCGGCCCGCGCGTCATGGGCGCCAACGGCGTCAGCCACCACGTCGTGGACGACGACCTGCAGGGCGTGCACGCCGTGCTGCGCTGGCTGGCCTACACGCCCGCGCGCGTGGGCGAGCTGCCGCCCACGCTGCGCGCGGCCGACCCCGTSGACCGCCGCGTGACCTACGCGCCGGCCGAGAACGAGAAGCTGGACCCGCGCCTGGCCGTGGCGGGCGGCGACGCGCCCGAGCCCGTGACGGGCCTCTTCGACCGCGGCAGCTGGACCGAGGCGCAGGCGGGCTGGGCACAGACCGTGGTCACGGGGCGCGCGCGCCTGGGCGGCATCCCCGTGGGCGTGGTGGCCGTGGAGGTGAACGCGGTGTCGCTGCACATCCCCGCCGACCCGGGCATGCCCGACTCGGCCGAGCGCACCATCCCGCAGGCGGGCCAGGTCTGGTTCCCGGACTCAGCGCTCAAGACCGCGCAGGCGATCGAGGAGTTTGGCCTGGAGGGCCTGCCCCTCTTCATCCTGGCCAACTGGCGCGGCTTCTCGGGCGGGCAGCGCGACCTGTTCGAGGGCGTGCTGCAGGCGGGCAGCCAGATCGTGGAGATGCTCCGCACCTACCGCCGCCCGGTSACCGTCTACCTGGGCCCGGCTGCGAGCTGCGCGGCGGCGCCTGGGTCGTGCTCGACTCCCATCAACCCGGCCAGCATCGAGATGTACGCCGACCCCACCGCGCAGGGCGCCGTGCTCGAGCCGCAGGGTGTCGTGGAGATCAAGTTCAGGACCCCAGACCTCCTCGCCGCCATGCACCGGCTGGACGAAAAGATCATCGCGCTCAAGTCGGACGACTCGCCCAGCGCGCTGGCGGCCATCAAGGCGCGAGAAAGCGAGCTGCTGCCCGTCTACAGCCAGGTCGCGCACCAGTTCGCGCAGATGCACGACGGCCCCGTGCGCATGCTCGCCAAGGGCGTGCTGCGCGGCATCGTGCCCTGGTCGGCCGCGCGCGCCTTCCTGGCCACGCGCCTGCGCCGGCGCCTGGCCGAGGAGGCGCTGCTTCGCCAGATCGCGGCGGCCGACGCGTCCGTCGAGCACGCCGACGCGCTGGCCATGCTGCGCTCCTGGTTCCTCAGCTCGCCGCCCACGGGCGGCGCGCCCGGCGCGCCCGGCGCGCTCGGCGCGCTGCTCAAGGAGACGGTCGTCGCGCCGCCCGACGCGGGCGAGGCGCCGCTGGCGCTCTGGCAGGACGACCTCGCCTTCCTGGACTGGAGCGAGGCCGAGGCCGGCGCCTCGCGCGTCGCGCTCGAGCTCAAGAGCCTGCGCGTCAACGTCGCCATGCGCAGCGTCGACAGACTGTGCCAGACCCCGGAGGGCACCGCCGGGCTCGTCAAGGGACTGGACGAGGCCATCAAGTCCAACCCGTCCCTCCTCCTCTGCCTCCGCTCGCTGGTCAAGCCGTAGPrototheca moriformis Homomeric Acetyl-CoA Carboxylase, ProteinSEQ ID NO: 263MTVANPPEAPFDSEGSSLAPDNGSSKPTKLSSTRSLLSISYRELSRSNQQSRSFTSRGVPGRTDVSDELERRILEWQGDRAIHSVLVANNGLAAVKFIRSIRSWSYKTFGNERAVKLIAMATPEDMRADAEHIRMADQFVEVPGGKNVQNYANVGLITSVAVRTGVDAVDCGGQIREVRTGVDAVWPGWGHASEEPELPESLGATPSQIREVGPPAGPMAALGDKVGSTILAQAAGVPTLAWSGSGVSIAYADCPRGEIPPEVYRRACIDSLEAALACCERIGYPVMLKASWGGGGKGIRKVLSADEVKLAYTQVCGEVPGSPVFAMKLAPQSRHLEVQLLCDAHGQVCSLYSRDCSVQRRHQKVVEEGPVTAAPPEVLEGMERCARSLARAVGYVGAATVEYLYMVETREYCFLELNPRLQVEHPVTEMITGVNIPAAQLLVAAGVPLHRIPDIRRLYRAPLAEDGPIDFEDDAARLPPNGHVLAVRVTAENAHDGFKPTSGAIEEISFRSTPDVWGYFSVKGGSAVHEFADSQFGHLFARGDSREAAVRAMVVALKEIKIRGEIQTPVDYVARMIQTDDFLGNRHHTGWLDARIAAQVGAERPPWFLVVIAGAVLRATRAVNERAAAFLDHLRKGQLPPSEVPLTRVAEEFVVDGTKYAVDVTRTGPQAYRVALRDPEGDGAPGHARRESLASAAGAASSVDVIARTLQDGGLLLQVRRARARAAQRGGGAGHAPGHRLGHVPAQQRARPLAARGRQHGQARAPPRRRGRARARGRALRRGRGHEDDDDAARAPRPARCAGRWPRAPRWRRACCSRAWRKLGRPSRRAPASPSAAPSPTWARPRPTLTAWPRATAPRSRPRRTCSTASRATCPRSSPRCWPRWTTPRSPSSCWTRRWPWSRRGCRPRWPRAWRPRLAEGAAEMAGELSDDEQDAALAAPGRRAKGGASTRASWSSAATRRRACSRASSRRAAGARRWPRRLLLERYLDVEERFESGGAKTDQEVIDGLRGTHSADPGKVLEIVVAHQSAGPRADLVHRLLNALVLPSPEAYRPILRRLAALTGAGSAAPAQRARRLLEHSLLGELRVLVAARAVRPWTWFSDAALRGLCLGESPGEPVSPRAELASALAAESSVASPTSRRSTLAEGLYVGLGNLAAAAASSVEARMAMLVEAPAAVDDALATLLDHPDPVLQRRALSTYVRRIYFPGVLHEPQVVGRRAGARSVRVWATRGAPGAMAPRPSRTSMAGLAPGTLHVVLTAEGAAALQLDAAAQAALGTLDVSGYVAPEHQQQPGLPDASVVAASAAAAVSALAPALREAGYSAVSFLTKRGGVEPLRVVFYDGTSSGSAAAPDSSSTQTGHRPAGPWTPCSARSSRRRPRRWSWPVWPAARAPRTTPRATVAHVDRGRARRQAQRGAAPHLFARRGALAGPAGAAERGLRGNGPAVAAAALAELEQTVEAAAAELERLGKGRVGGASSRLDAPCLSVLMLAAAGRVRAARRERRRRARAGRAAAIASRHVAALRRAALAQWEVCLRTGSGGASHREGGWRVVVSSPTGHEAGEAFVDVYREAADGTLRAVNPSLRAPGPLDGQSVLAPYPTLAPLQQRRLTARRHKTTYAYDFPAVFEDALRSIWLQRAVELGATGLEAAREDLLPPGTAWSRPELVLDTEEAIYENGAAHIRLTDRAPSMNDLGVVAWRLTLATPECPRGRAVVAIANDITYNSGSFGPREDAFFKAATEYALAERLPVVYLAANSGARVGLAEEVKRCLRVEWSVPGDPTKGHKYLYLDDEDYRSITGRAAGRTLPVSCSAKVGADGRTRHVLHDVIGLEDGLGVECLSGSGAIAGAFARAFREGFTVTLVSGRTVGIGAYLARLGRRCVQRREQPIILTGFAALNKLLGREVYTSQQQLGGPRVMGANGVSHHVVDDDLQGVHAVLRWLAYTPARVGELPPTLRAADPVDRRVTYAPAENEKLDPRLAVAGGDAPEPVTGLFDRGSWTEAQAGWAQTVVTGRARLGGIPVGVVAVEVNAVSLHIPADPGMPDSAERTIPQAGQVWFPDSALKTAQAIEEFGLEGLPLFILANWRGFSGGQRDLFEGVLQAGSQIVEMLRTYRRPVTVYLGPAASCAAAPGSCSTPINPASIEMYADPTAQGAVLEPQGVVEIKFRTPDLLAAMHRLDEKIIALKSDDSPSALAAIKARESELLPVYSQVAHQFAQMHDGPVRMLAKGVLRGIVPWSAARAFLATRLRRRLAEEALLRQIAAADASVEHADALAMLRSWFLSSPPTGGAPGAPGALGALLKETVVAPPDAGEAPLALWQDDLAFLDWSEAEAGASRVALELKSLRVNVAMRSVDRLCQTPEGTAGLVKGLDEAIKSNPSLLLCLRSLVKP*Prototheca moriformis Nitrogen Response Regulator (NRR1) SEQ ID NO: 264ATGGAGGAGCCGGACCAACAGGACCCTGCGGGCTCCGGGACCACCCAGGCCGAGGCTGAAAAGAGGAGTGCATCCCCAAAGCCCGTCAGCGAGGCCACCAGCGACGGCAAGGCCTCCTCCGTGGCCACGGCGGCCGAAGCCAAGCCCGAAAGCTCTCCAGGCGAGGATGTGGCCGCCCTCTACCCCGGCATCCCCGTCCTGCCGGACCTGGGCCGGTTGCGGGACAACCGGCGCGACGTGCCCCTGACCTGCCAGGTGGTGGGCTGCGGCGAGAGCCTCAGCGGGCACGCCGAGTACTACCGGCGGTACCGCGTCTGCAAGCGCCACCTCAAGGGCTCGGCGCTGATCGTGGACGGGGTGCCGCAGCGATTCTGCCAGCAGTGCGGTCGCTTCCACCTGCTGGAGGCCTTTGACGGCGAGAAGAGAAACTGCCGCGCCCGGCTGGAGGAGCACAACTCGCGGCGGCGAAAGGTCATGGCGCCGGGGGGCGGCGGCCAGGTGACGGGCGCGCGCGCGACGCGCCGGGAGTCGGAGCGGCACGGCCGCGGCATGGACCTGCGCGGCGCCGGGCTCGAGGTCGCTCGGTCGCCCGPrototheca moriformis Nitrogen Response Regulator (NRR1), proteinSEQ ID NO: 265MEEPDQQDPAGSGTTQAEAEKRSASPKPVSEATSDGKASSVATAAEAKPESSPGEDVAALYPGIPVLPDLGRLRDNRRDVPLTCQVVGCGESLSGHAEYYRRYRVCKRHLKGSALIVDGVPQRFCQQCGREHLLEAFDGEKRNCRARLEEHNSRRRKVMAPGGGGQVTGARATRRESERHGRGMDLRGAGLEVARSPPrototheca moriformis Monoacylglycerol acyltransferase (MGAT1)SEQ ID NO: 266CGCAAAGGGTGTTCAGTGGTCGATGGGGACGAACTTGACGTCCTCGTAGCCTGGGAAGCTGGGCGCGTAGCGTGCGAAGAGGTCCTCCAGCGAGGCGTAGAACCGCGCGTGGACGTCGTCGATCTGGGCCTGCGAGGGGCCCTCTGGCGCCACCTTTGTCGCCGGGAGGATGGGCTTGCCGATCACAAACCTGAGTCCTGTGGGGCGGGGGAAGGGCGACACCTGGTAGCGCCCGCCGATGAGGTAGGGCACGGGGAAGCCCAGGCGCTTGTAGGTCCAGCGCAGCATGCCCGGCAGGTTGACCAGGTTTTGGAGCGAGTCGACCTCGCCCAGCGCCAGCACCGGCACGAGCGCGGCCTCGCTCTGCAGCGCCAGCCGCACAAAGCCCTTGTGGCTGCGGAACACGCAAAACTCCCGCGTCGGCCGCGCGATGCGGTGCGTGTGCACCAGCTCGGCCTGGCCACCGGGGACGAGCAGCACGGCGCCCTTCTCGCGCAGCGCGCGGAGGAAGGATCGCCGCGAGACGACGCGCAGGCCGAGCCAGGCCAGCAGGTCGCGCAGCAAGGGCACCGCAAACACGATCGAGGCGCAGAGCGTCACGGGGCGGATGCCGGGGAGCAGGCGCCGGAAGCCGGGCAGCAGCGGCAGGTAGCCGGCCGCCAGCGGGTACAGACCGTGCACGGCAAAGCCAAAGACGTGGCGGCGCTCGGGGTCGTACTGGTCCGCCTCGCCGTCGCGCAGGATCTCCAGCGACCCTACCCAGGCCGGGAGCACCTCCTCCGACCGCCGCCCAAACCAGTCCCGGAACAGCGGCCACTCCCGCCGGCCTGTCTCGCCAAAGGGCCCGCCAATCAGGGTGATGCTGTAGTACACAAAGGCCGAAGCGAGCAGGAGCGTCAGGGCAATCTCCAGCGGAAAGAGAAGAAAGTAGAGCGCAATGATTGTGCACAGCAACTGGACCGGGGCGTAGAGGAGGGTGATGACAAGGGCGTCGGAGAGCCGGGTCCGTAGGTCGGTCGGCCGCAGCGCGCGCTCCGAGAGCGACGCGCTGAGTCCGCGCACGAGCTCCATGGTCCGCACGGGGCCCTTGTACAGCAACAGCGAGCCGGCCAGCACCAGGTGGGAGAAGACCATGAAGATGCCTGCGGTCTGGGCAAAGCGCTGCGCGCTGTGCACGATGCCGATGGTGCTGTGGGCCATGAGGTAGATGATGAGGCTCAGCCCCGCGGAGTAGAGCGCCCACCCCAGGGCCTGCGTGGCCACGAACCAGCCGCCGCCCTTGAAGGGCTGGAAAAAGTGCCATGCGGCCGGCGCGGCCTCATCGCCGCCCTCGGAGTCGCTCTCGCTGCCGGGCTCGGACTTGCGATGCACGTGCCGCAGGTGGCCCGCCAGCCCGTAGGTCACAAAGGCGGCCATGGTCAAGCATGCGCCCGAGAGCGCCAGGTACGCCAGCCGCACGGGCGCGTCCTCCAGCAGGTTGGCGCCCAGGTGCAGCAGCGCGGCGCCCAGCGCGACCCAGAGCGACCCCATCCCGATCACGACGTACGAGGCCGCGGCGGACGACGGCAGCGACTTGACGTCCCACAAGACATCGGTGTGGCCCTGCTGGAGACCCGCGAGGATGCGCATGGGCTTGGCCGCGGGGTCAAAGACCAGCACGGCAGACACCTGGAAGAGCTGGGAGGACACGGCTCCCACGGCCGCGGCGGCCACCAGCAGCTCCACCGTCAGGCTAGACATCCAATCGTGCCGCGCGGCCGACAGAATGGGCACCACTACGGCGCAGCTCAGGAGCCAAGCTGAGAAGGACATGGCCTGGAAGACGAGGGTGCGTACCCCGCCCATCGTGCCCCAGCTCCACCCGGGCCGGACCTGGGTCTTGCCCCTGCCCAGCTGCACGGCGCGGTGCAGCCATCGCGCCCCGCCAACGTAGGTGAGGGGCGTGGACACCGCGAACACCACCCAGCAGGCCGCCAGGCAAATGACCACGTGGTCGCCGGAGAGGACCGACTCCGAGAGGGAGTGGCTCACCGACGTCATAGCAAGGGCCCTCGTCCGTCAGACAGACTCATTCGATGCACATCCCTGCTCCCGCGGTGGGAATGAGGTTGGGAAAAAGATGGGATCGACCTGGGAATGGAAGCGAACTGCCAGGAAGGCATCGTCACCGGGGGTTCCGACAACCATATCGTCATTGATGCATCGGGGCCTCCGGGGAGGCGTCTGCCGCCGGACATGGGAGCGATCAGGCGACCGGAGTGTTTAAGCGAGCACACGACCAGCACCGTCGTTGAGCCTCGAAAGTGATGCAAAPrototheca moriformis Monoacylglycerol acyltransferase (MGAT1), proteinSEQ ID NO: 267MTSVSHSLSESVLSGDHVVICLAACWVVFAVSTPLTYVGGARWLHRAVQLGRGKTQVRPGWSWGTMGGVRTLVFQAMSFSAWLLSCAVVVPILSAARHDWMSSLTVELLVAAAAVGAVSSQLFQVSAVLVFDPAAKPMRILAGLQQGHTDVLWDVKSLPSSAAASYVVIGMGSLWVALGAALLHLGANLLEDAPVRLAYLALSGACLTMAAFVTYGLAGHLRHVHRKSEPGSESDSEGGDEAAPAAWHFFQPFKGGGWEVATQALGWALYSAGLSLITYLMAHSTIGIVHSAQRFAQTAGIFMVFSHLVLAGSLLLYKGPVRTMELVRGLSASLSERALRPTDLRTRLSDALVITLLYAPVQLLCTIIALYFLLFPLEIALTLLLASAFVYYSITLIGGPFGETGRREWPLERDWFGRRSEEVLPAWVGSLEILRDGEADQYDPERRHVFGFAVHGLYPLAAGYLPLLPGFRRLLPGIRPVTLCASIVFAVPLLRDLLAWLGLRVVSRRSFLRALREKGAVLLVPGGQAELVHTHRIARPTREFCVERSHKGEVRLALQSEAALVPVLALGEVDSLQNLVNLPGMLRWTYKRLGFPVPYLIGGRYQVSPFPRPTGLREVIGKPILPATKVAPEGPSQAQIDDVHARFYASLEDLFARYAPSFPGYEDVKFVPIDH *Prototheca moriformis Cellulase, Endoglucanase (EG1) SEQ ID NO: 268CTCCTCCCAGCCACCGTCCACCAAATCCCACCTCTCGCCCGCTCAGACGTTGATGTAGGTGCCGCAGATGTCGGAGCGGCGGAAGATGCCGTACTGCTGCAGGCACTGCTCCCAGAGCCCAGAGGGCAGCTTGCTGGCGCCGGCCAGGGCGCCGATCAGGCCCGCGTTGTTCTCCACGCCCACGTAGGCCGCGGTCGTGTCGCGCGTGTCCACAAAGTTGTCCGAGCTGGCCGTGCCGTACACGAGCGCGCCCGTGAGCGTGTGCGTGTCGGGGTCGGGCGACAGCAGACCGGTCACGCGGTTGCACACCTCTGGCTCGGCCGGGCAGGCCGCGTCGCGGTCCTGCGTGCGCTTGGGCGGGTTCTTGCCAAAGCCGACGACGAGCGAGCGCCCCGAGTCGCCCAGCACGTAGCGGATCTGGCTCAGGCCCCAGCACTGGTACTCAAACACGCGCGTCGACCCGGGGTCGTTCAGCGAGTCCGCGTAGGCCAGCGCCGCAAAGGCCGTGTTCATGGTGGAGCCCAGCGGCGCGCTCATGGGGTTGTAGGCGCGGCCCTTGAGCGTGTAGTTGGCCGCCAGGTCGGAGCAGATCCAGGACTTGAGGAACAGCTCCGACTGGCTCTGGAAGGTCACGCCGCCCGTCTCCTGCGCCAGCAGCACGTTGGTCGGCCAGAACACGTTGTCCCAGTCAAACGCGTACTTGAAGTCGCTCACCGACGACTCGTACTCCAGGTGGTTCACGTACTGGCTGTACATGTTGGTCAGGTACTGCTGCTCCCCCGTCGCCTTGTACATCCAGCTGCTCGACCACGCCAGGTCGTCGTAAAACGTGCTCGAGTTGTACACCTTGGAAATGTTCCAGTCCTGCACCGAGTAGTGGCCCAGGTCGGTGGAGGCGATGTCGTACACGGCCTTGGCCTTGGTCAGCAGCTCCGTCGCGTAGGCCGCGTCGCCGCTGGCGTTGAACACGATGGAGGCCGAGGCCAGCGCCGCCGCGACCTGGCCGCCCAGGTCGCTCGCGCCCTTGGCCATGTCCACCACGTACGCGGGCCGGTCCGTGGTGTAGTCCTCCAGACGGTACCAGTTGAGCTCCTCCTGCTGGATGTTGCCAATCCTGGACAGAATGTAGGTGGTGTTGCTCGTCGTCTGCACCGTCTTGAGCAGGTAGTCCGTGGACCAGCGCAGGTTGTCCAGCAGCGACGCGGAGCTGCCCGCGTCCCCGATGGCCTCGCCGTAGGTCTGGTAGGCCCAGGACATGAGCGTCGCCGTAAAGGCGATGGGCAGCGTGGCCTTGAAGTTGCCGGCCGCCATGCCCGTCACGTAGCCGCCCTCCAGGTCCCAGGTCGCGCCCAGCTTGACCGCCTGCGGCACCGACTTGGTCGACGAGGAGGGGCTGATCGTGGAAGGCGGCGGCAGGGTGGGGATGAGCGACGAAGGCGAGGAGCCCGGGGTGCTGGGCGACGAGCCCGCCACGGGCGACCACACCAGGTTGGCAAACGGPrototheca moriformis Cellulase, Endoglucanase (EG1), proteinSEQ ID NO: 269PFANLVWSPVAGSSPSTPGSSPSSLIPTLPPPSTISPSSSTKSVPQAVKLGATWDLEGGYVTGMAAGNFKATLPIAFTATLMSWAYQTYGEAIGDAGSSASLLDNLRWSTDYLLKTVQTTSNTTYILSRIGNIQQEELNWYRLEDYTTDRPAYVVDMAKGASDLGGQVAAALASASIVFNASGDAAYATELLTKAKAVYDIASTDLGHYSVQDWNISKVYNSSTFYDDLAWSSSWMYKATGEQQYLTNMYSQYVNHLEYESSVSDFKYAFDWDNVEWPTNVLLAQETGGVTFQSQSELFLKSWICSDLAANYTLKGRAYNPMSAPLGSTMNTAFAALAYADSLNDPGSTRVFEYQCWGLSQIRYVLGDSGRSLVVGFGKNPPKRTQDRDAACPAEPEVCNRVTGLLSPDPDTHTLTGALVYGTASSDNFVDTRDTTAAYVGVENNAGLIGALAGASKLPSGLWEQCLQQYGIFRRSDICGTYINV* P. moriformis KAS II SEQ ID NO: 270MQTAHQRPPTEGHCFGARLPTASRRAVRRAWSRIARAAAAADANPARPERRVVITGQGVVTSLGQTIEQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALESAGLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMDIGFMGPNYSISTACATGNYCILGAADHIRRGDANVMLAGGADAAIIPSGIGGFIACKALSKRNDEPERASRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATILAELVGGAATSDAHHMTEPDPQGRGVRLCLERALERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVIPQDSLRINSTKSMIGHLLGGAGAVEAVAAIQALRTGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLDVVLSNSFGEGGHNSCVIERKYDE P. moriformis stearoyl ACP desaturase SEQ ID NO: 271MASAVTFACAPPRGAVAAPGRRAASRPLVVRAVASEAPLGVPPSVQRPSPVVYSKLDKQHRLTPERLELVQSMGQFAEERVLPVLHPVDKLWQPQDFLPDPESPDFEDQVAELRARAKDLPDEYFVVLVGDMITEEALPTYMAMLNTLDGVRDDTGAADHPWARWTRQWVAEENRHGDLLNKYCWLTGRVNMRAVEVTINNLIKSGMNPQTDNNPYLGFVYTSFQERATKYSHGNTARLAAEHGDKNLSKICGLIASDEGRHEIAYTRIVDEFFRLDPEGAVAAYANMMRKQITMPAHLMDDMGHGEANPGRNLFADFSAVAEKIDVYDAEDYCRILEHLNARWKVDERQVSGQAAADQEYVLGLPQRFRKLAEKTAAKRKRVARRPVAFSWISGREIMV Prototheca moriformis d12 desaturase allele 1SEQ ID NO: 272MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGVMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVEHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVEVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMENVASRPYPRFANHFDPWSPIFSKRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYLIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDNILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHKPrototheca moriformis delta 12 desaturase allele 2 SEQ ID NO: 273MAIKTNRQPVEKPPFTIGTLRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPTWVKYGIMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVEHSLLLVPYYSWKHSHRRHHSNTGCLDKDEVEVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMENVASRPYPRFANHFDPWSPIFSKRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYMIVNMWLVLITLLQHTHPALPHYFEKDWDWLRGAMATVDRSMGPPFMDSILHHISDTHVLHHLFSTIPHYHAFEASAAIRPILGKYYQSDSRWVGRALWEDWRDCRYVVPDAPEDDSALWFHK

1. A recombinant nucleic acid comprising a coding sequence that encodesa Prototheca lipid biosynthesis protein.
 2. The recombinant nucleic acidof claim 1 wherein the lipid biosynthesis protein is selected from aprotein in Table
 1. 3. The recombinant nucleic acid of claim 2 whereinthe protein has at least one point mutation in comparison to a proteinin Table
 1. 4. The recombinant nucleic acid of claim 1 wherein theprotein has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequenceidentity to a protein in Table
 1. 5. The recombinant nucleic acid ofclaim 1, wherein the coding sequence is in operable linkage with apromoter.
 6. The recombinant nucleic acid of claim 1, wherein the codingsequence is in operable linkage with an untranslated control element. 7.The recombinant nucleic acid of claim 1, wherein the coding sequence isin operable linkage with a targeting sequence.
 8. The recombinantnucleic acid of claim 7, wherein the targeting sequence is a transitpeptide selected from the group of plastidial targeting sequence andmitochondrial targeting sequence.
 9. The recombinant nucleic acid ofclaim 1, wherein the nucleic acid is a DNA molecule.
 10. The recombinantnucleic acid of claim 1 that encodes a functional Prototheca lipidbiosynthesis protein.
 11. The recombinant nucleic acid of claim 3wherein the protein is diacylglycerol diacyltransferase (DGAT) having atleast one point mutation.
 12. The recombinant nucleic acid of claim 1that further encodes a sucrose invertase.
 13. The recombinant nucleicacid of claim 1 that further encodes an inhibitory RNA that suppressesexpression of a Prototheca lipid biosynthesis gene.
 14. An expressioncassette comprising a recombinant nucleic acid of claim
 1. 15. Agenetically engineered microbial cell transformed with a recombinantnucleic acid of claim
 1. 16-29. (canceled)
 30. A method for obtainingmicrobial oil comprising culturing a genetically engineered Protothecacell of claim 15 under conditions such that oil is produced.
 31. Amicrobial oil produced by the method of claim
 30. 32. A geneticallyengineered cell of the genus Prototheca wherein the activity of one ormore endogenous lipid biosynthesis gene selected from the genes listedin Table 1 has been attenuated. 33-39. (canceled)
 40. A method forobtaining microbial oil comprising culturing a genetically engineeredPrototheca cell of claim 32 under conditions such that oil is produced.41. A microbial oil produced by the method of claim 40.